Human Anti-Tau Antibodies

ABSTRACT

Provided are novel human tau-specific antibodies as well as fragments, derivatives and variants thereof as well as methods related thereto. Assays, kits, and solid supports related to antibodies specific for tau are also disclosed. The antibody, immunoglobulin chain(s), as well as binding fragments, derivatives and variants thereof can be used in pharmaceutical and diagnostic compositions for tau targeted immunotherapy and diagnosis, respectively.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/391,751, filed Oct. 11, 2010, and EP Application No. 10013494.9,filed Oct. 11, 2010, each of which is herein incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention generally relates to novel tau-specific bindingmolecules, particularly human antibodies as well as fragments,derivatives and variants thereof that recognize the tau protein,including pathologically phosphorylated tau and aggregated forms of tau.In addition, the present invention relates to pharmaceutical anddiagnostic compositions comprising such binding molecules, antibodiesand mimics thereof valuable both as a diagnostic tool to identify tauand toxic tau species in plasma and CSF and also in passive vaccinationstrategies for treating neurodegenerative tauopathies such asAlzheimer's disease (AD), amyotrophic lateralsclerosis/parkinsonism-dementia complex (ALS-PDC), argyrophilic graindementia (AGD), British type amyloid angiopathy, cerebral amyloidangiopathy, corticobasal degeneration (CBD), Creutzfeldt-Jakob disease(CJD), dementia pugilistica, diffuse neurofibrillary tangles withcalcification, Down's syndrome, frontotemporal dementia, frontotemporaldementia with parkinsonism linked to chromosome 17 (FTDP-17),frontotemporal lobar degeneration, Gerstmann-Sträussler-Scheinkerdisease, Hallervorden-Spatz disease, inclusion body myositis, multiplesystem atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C),non-Guamanian motor neuron disease with neurofibrillary tangles, Pick'sdisease (PiD), postencephalitic parkinsonism, prion protein cerebralamyloid angiopathy, progressive subcortical gliosis, progressivesupranuclear palsy (PSP), subacute sclerosing panencephalitis, tangleonly dementia, multi-infarct dementia and ischemic stroke.

Background Art

Protein accumulation, modifications and aggregation are pathologicalaspects of numerous neurodegenerative diseases. Pathologically modifiedand aggregated tau including hyperphosphorylated tau conformers are aninvariant hallmark of tauopathies and correlate with disease severity.

Tau is a microtubule-associated protein expressed in the central nervoussystem with a primary function to stabilize microtubules. There are sixmajor isoforms of tau expressed in the adult human brain, which arederived from a single gene by alternative splicing. Under pathologicalconditions, the tau protein becomes hyperphosphorylated, resulting in aloss of tubulin binding and destabilization of microtubules followed bythe aggregation and deposition of tau in pathogenic neurofibrillarytangles. Disorders related to tau—collectively referred to asneurodegenerative tauopathies—are part of a group of protein misfoldingdisorders including Alzheimer's disease (AD), progressive supranuclearpalsy, Pick's disease, corticabasal degeneration, FTDP-17 among others.More than 40 mutations in tau gene have been reported to be associatedwith hereditary frontotemporal dementia demonstrating that tau genemutations are sufficient to trigger neurodegeneration (Cairns et al.,Am. J. Pathol. 171 (2007), 227-40). Studies in transgenic mice and cellculture indicate that in AD, tau pathology may be caused by apathological cascade in which Aβ lies upstream of tau (Götz et al.,Science 293 (2001), 1491-1495). Other finding however point to adual-pathway model where both cascades function independently of eachother (van de Nes et al., Acta Neuropathol. 111 (2006), 126-138).Immunotherapies targeting the beta-amyloid peptide in AD have producedencouraging results in animal models and shown promise in clinicaltrials. More recent autopsy data from a small number of subjectssuggests that clearance of beta-amyloid plaques in patients withprogressed AD may not be sufficient to halt cognitive deterioration,emphasizing the need for additional therapeutic strategies for AD(Holmes et al., Lancet 372 (2008), 216-223; Boche et al., ActaNeuropathol. 120 (2010), 13-20). In the wake of the success ofAbeta-based immunization therapy in transgenic animal models, theconcept of active immunotherapy was expanded to the tau protein. Activevaccination of wild type mice using the tau protein was however found toinduce the formation of neurofibrillary tangles, axonal damage andmononuclear infiltrates in the central nervous system, accompanied byneurologic deficits (Rosenmann et al., Arch Neurol. 63 (2006),1459-1467). Subsequent studies in transgenic mouse lines using activevaccination with phosphorylated tau peptides revealed reduced brainlevels of tau aggregates in the brain and slowed progression of behaviorimpairments (Sigurdsson, J. Alzheimers. Dis. 15 (2008), 157-168; Boimelet al., Exp. Neurol. 224 (2010), 472-485). These findings highlight thepotential benefit but also the tremendous risks associated with activeimmunotherapy approaches targeting tau. Novel therapeutic strategies areurgently needed addressing pathological tau proteins with efficaciousand safe therapy.

Passive immunization with human antibodies derived from healthy humansubjects which are evolutionarily optimized and affinity matured by thehuman immune system would provide a promising new therapeutic avenuewith a high probability for excellent efficacy and safety.

BRIEF SUMMARY OF THE INVENTION

The present invention makes use of the tau-specific immune response ofhealthy human subjects for the isolation of natural anti-tau specifichuman monoclonal antibodies. In particular, experiments performed inaccordance with the present invention were successful in the isolationof monoclonal tau-specific antibodies from a pool of healthy humansubjects with no signs of a neurodegenerative tauopathy.

The present invention is thus directed to human antibodies,antigen-binding fragments and similar antigen-binding molecules whichare capable of specifically recognizing tau. By “specificallyrecognizing tau”, “antibody specific to/for tau” and “anti-tau antibody”is meant specifically, generally, and collectively, antibodies to thenative form of tau, or aggregated or pathologically modified tauisoforms. Provided herein are human antibodies selective forfull-length, pathologically phosphorylated and aggregated forms.

In a particular embodiment of the present invention, the human antibodyor antigen-binding fragment thereof demonstrates the immunologicalbinding characteristics of an antibody characterized by the variableregions V_(H) and/or V_(L) as set forth in FIG. 1.

The antigen-binding fragment of the antibody can be a single chain Fvfragment, an F(ab′) fragment, an F(ab) fragment, and an F(ab′)₂fragment, or any other antigen-binding fragment. In a specificembodiment, infra, the antibody or fragment thereof is a human IgGisotype antibody. Alternatively, the antibody is a chimeric human-murineor murinized antibody, the latter being particularly useful fordiagnostic methods and studies in animals.

Furthermore, the present invention relates to compositions comprisingthe antibody of the present invention or active fragments thereof, oragonists and cognate molecules, or alternately, antagonists of the sameand to immunotherapeutic and immunodiagnostic methods using suchcompositions in the prevention, diagnosis or treatment of a tauopathy,wherein an effective amount of the composition is administered to apatient in need thereof.

Naturally, the present invention extends to the immortalized human Bmemory lymphocyte and B cell, respectively, that produces the antibodyhaving the distinct and unique characteristics as defined below.

The present invention also relates to polynucleotides encoding at leasta variable region of an immunoglobulin chain of the antibody of theinvention. In one embodiment, said variable region comprises at leastone complementarity determining region (CDR) of the V_(H) and/or V_(L)of the variable region as set forth in FIG. 1.

Accordingly, the present invention also encompasses vectors comprisingsaid polynucleotides and host cells transformed therewith as well astheir use for the production of an antibody and equivalent bindingmolecules which are specific for tau. Means and methods for therecombinant production of antibodies and mimics thereof as well asmethods of screening for competing binding molecules, which may or maynot be antibodies, are known in the art. However, as described herein,in particular with respect to therapeutic applications in human theantibody of the present invention is a human antibody in the sense thatapplication of said antibody is substantially free of an immune responsedirected against such antibody otherwise observed for chimeric and evenhumanized antibodies.

Furthermore, disclosed herein are compositions and methods that can beused to identify tau in samples. The disclosed anti-tau antibodies canbe used to screen human blood, CSF, and urine for the presence of tau insamples, for example, by using ELISA-based or surface adapted assay. Themethods and compositions disclosed herein can aid in neurodegenerativetauopathies such as Alzheimer's disease diagnosis and can be used tomonitor disease progression and therapeutic efficacy.

Hence, it is a particular object of the present invention to providemethods for treating, diagnosing or preventing a neurodegenerativetauopathy such as Alzheimer's disease, amyotrophic lateralsclerosis/parkinsonism-dementia complex, argyrophilic grain dementia,British type amyloid angiopathy, cerebral amyloid angiopathy,corticobasal degeneration, Creutzfeldt-Jakob disease, dementiapugilistica, diffuse neurofibrillary tangles with calcification, Down'ssyndrome, frontotemporal dementia, frontotemporal dementia withparkinsonism linked to chromosome 17, frontotemporal lobar degeneration,Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease,inclusion body myositis, multiple system atrophy, myotonic dystrophy,Niemann-Pick disease type C, non-Guamanian motor neuron disease withneurofibrillary tangles, Pick's disease, postencephalitic parkinsonism,prion protein cerebral amyloid angiopathy, progressive subcorticalgliosis, progressive supranuclear palsy, subacute sclerosingpanencephalitis, tangle only dementia, multi-infarct dementia andischemic stroke. The methods comprise administering an effectiveconcentration of a human antibody or antibody derivative to the subjectwhere the antibody targets tau.

Further embodiments of the present invention will be apparent from thedescription and Examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino acid and nucleotide sequences of the variable region, i.e.heavy chain and kappa/lambda light chain of human antibodies NI-105-4E4(A), NI-105-24B2 (B) and NI-105.4A3 (C). Framework (FR) andcomplementarity determining regions (CDRs) are indicated with the CDRsbeing underlined. Due to the cloning strategy the amino acid sequence atthe N-terminus of the heavy chain and light chain may potentiallycontain primer-induced alterations in FR1, which however do notsubstantially affect the biological activity of the antibody. In orderto provide a consensus human antibody, the nucleotide and amino acidsequences of the original clone were aligned with and tuned inaccordance with the pertinent human germ line variable region sequencesin the database; see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/)hosted by the MRC Centre for Protein Engineering (Cambridge, UK). Thoseamino acids, which are considered to potentially deviate from theconsensus germ line sequence due to the PCR primer and thus have beenreplaced in the amino acid sequence, are indicated in bold.

FIG. 2: ELISA plates were coated with recombinant human tau (isoformhTau40) at 1 μg/ml and incubated with the indicated concentrations ofNI-105.4E4 antibody. Recombinant human derived antibody NI-105.4E4 bindsto recombinant tau with high affinity at 2 nM EC₅₀.

FIG. 3: PHFTau and recombinant hTau40 were resolved by gradient SDS-PAGEfollowed by Western Blot analysis. Blots were incubated with primaryantibodies NI-105.4E4 (human) or mouse monoclonal Tau12 antibody,followed by HRP-conjugated secondary antibodies. Recombinant human tauantibody NI-105.4E4 binds to recombinant hTau40 as well as topathologically modified tau isoforms (PHFTau) extracted from AD brain onWestern blot analysis.

FIGS. 4A, 4B, 4C, 4D and 4E: Mapping of the NI-105.4E4 binding epitopeon hTau40. PepSpot (JPT) technology: Two groups of adjacent peptidespots (peptide 83, 84 and 85; peptide 96 and 97) were specificallyidentified by NI105.4E4 (FIG. 4A and FIG. 4A′), when compared to thedetection antibody only (FIG. 4B). The HRP-conjugated goat anti-humanIgG detection antibody alone produces a strong signal on single spot(peptide 50) but does not detect peptides 83, 84, 85, 96 and 97. Alaninescanning: (FIG. 4C) Spots #35-50 and #51-68 contain the originalpeptides (spots #35 and #51) and their substituted variants (#36-50 and#52-68) (FIGS. 4D and 4E) Amino acid sequence of the original andsubstituted peptides (#35-50 and #51-68). Alanine scan suggests residuesV339, E342, D387, E391 and K395 contribute to NI-105.4E4 binding.

FIG. 5: Confirmation that the human recombinant NI-105.4E4 antibodybinds specifically to a tau peptide corresponding to amino acids 333-346of hTau40.

FIG. 6: NI-105.4E4 binds to neurofibrillary tangles (NFT), dystrophicneurites and neuropil threads in AD brain and human TauP301L expressingmice. NI105-4E4 staining identifies NFTs and neuropil threads in ADbrain (A), with no significant binding to tau in the brain of healthycontrol subject (B). In TauP301L transgenic mouse (E-I) NI-105.4E4 bindsstrongly to the pathological tau resembling NFT (E, F and H), neuropilthreads (E and G) and dystrophic neurites (E and H). In addition,NI-105.4E4 also identifies tau aggregates at pre-tangle stage (I).NI-105.4E4 binds to NFT, dystrophic neurites and neuropil threads intransgenic mouse expressing human APP with the Swedish and the Arcticmutation and TauP301L; the arrow marks a beta-amyloid plaque, surroundedby dystrophic neurites recognized by NI-105.4E4 (J). Secondary antibodyonly does not give signal both in human AD (C) and healthy control (D).

FIG. 7: ELISA plates were coated with recombinant human tau (hTau40) at3 μg/ml and incubated with the indicated concentrations of NI-105.24B2antibody. Recombinant human derived antibody NI-105.24B2 binds to hTau40with high affinity at 6 nM EC₅₀.

FIG. 8: PHFTau and recombinant hTau40 were resolved by gradient SDS-PAGEfollowed by Western Blot analysis. Blots were incubated overnight withprimary antibodies NI-105.24B2 (human), followed by HRP-conjugatedanti-human IgG. Recombinant human tau antibody NI-105.24B2 binds torecombinant hTau40 as well as to pathologically modified tau isoforms(PHFTau) extracted from AD brain on Western Blot analysis.

FIG. 9: Tissue amyloid plaque immunoreactivity (TAPIR) assay—Serumisolated from elderly subjects was added to histological AD brainsections. As a comparison an immunohistological staining with thecommercially available AT100 anti-phospho-tau antibody was performed.Neurofibrillary tangles are stained in the control staining with AT100anti-phospho-tau antibody when subjected to isolated sera, showing thepresence of neurofibrillary tangles-reactive antibody species in thetested sera.

FIG. 10: Recombinant human antibody NI-105.4A3 specifically binds tohuman tau by ELISA. No binding is observed to BSA.

FIG. 11: The NI-105.4E4 and NI-105.4A3 epitopes and epitopes of commonlyused commercially available mouse monoclonal tau antibodies are shown.Human antibody NI-105.4E4 targets a unique epitope that comprises twolinear polypeptides, one of which is located in the microtubule bindingdomain (R4) of tau which is masked in physiologicalmicrotubule-associated tau. Tau-12 (Covance, Calif., U.S.A.), HT7, AT8,AT180 (Thermo Scientific, U.S.A.); PHF1 (Lewis et al., Science 293(2001), 1487-1491).

FIG. 12: ELISA plates were coated with recombinant human tau (hTau40, 1ug/ml), PHFTau (1:100) and control preparation (1:100), and incubatedwith indicated concentration of NI-105.4A3. 4A3 binds to rTau with 1.4nM EC50, to PHFTau with 1.2 nM EC50.

FIG. 13: Mapping of the NI-105.4A3 binding epitope on hTau40. (A)Schematic representation of the four overlapping hTau40 domains (domainI (AA 1-158), domain II (AA 136-258), domain III (AA 235-373), anddomain IV (AA 355-441)) used. (B) NI-105.4A3 binds only tau domain I andthe full length hTau40 polypeptide. (C) Western blot confirms thespecific bonding of NI-105.4A3 to tau domain I.

FIG. 14: NI-105.4A3 epitope mapping with PepSpot (JPT) technology (A andB) and alanine scanning (C).

FIG. 15: Binding of ch4E4 and variants to recombinant tau (ELISA).

FIG. 16: Human IgG levels in the plasma of mice followingintraperitoneal administration of 30 mg/kg 4E4 or 4A3 human anti-tauantibody.

FIG. 17: Human IgG levels in brain homogenate of mice followingintraperitoneal administration of 30 mg/kg 4E4 or 4A3 human anti-tauantibody.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Neurodegenerative tauopathies are a diverse group of neurodegenerativedisorders that share a common pathologic lesion consisting ofintracellular aggregates of abnormal filaments that are mainly composedof pathologically hyperphosphorylated tau in neurons and/or glial cells.Clinical features of the tauopathies are heterogeneous and characterizedby dementia and/or motor syndromes. The progressive accumulation offilamentous tau inclusions may cause neuronal and glial degeneration incombination with other deposits as, e.g., beta-amyloid in Alzheimer'sdisease or as a sole pathenogenic entity as illustrated by mutations inthe tau gene that are associated with familial forms of frontotemporaldementia and parkinsonism linked to chromosome 17 (FTDP-17). Because ofthe heterogeneity of their clinical manifestations a potentiallynon-exhaustive list of tauopathic diseases may be provided includingAlzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementiacomplex, argyrophilic grain dementia, British type amyloid angiopathy,cerebral amyloid angiopathy, corticobasal degeneration,Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillarytangles with calcification, Down's syndrome, frontotemporal dementia,frontotemporal dementia with parkinsonism linked to chromosome 17,frontotemporal lobar degeneration, Gerstmann-Sträussler-Scheinkerdisease, Hallervorden-Spatz disease, inclusion body myositis, multiplesystem atrophy, myotonic dystrophy, Niemann-Pick disease type C,non-Guamanian motor neuron disease with neurofibrillary tangles, Pick'sdisease, postencephalitic parkinsonism, prion protein cerebral amyloidangiopathy, progressive subcortical gliosis, progressive supranuclearpalsy, subacute sclerosing panencephalitis, tangle only dementia,multi-infarct dementia and ischemic stroke; see for a review, e.g., Leeet al., Annu. Rev. Neurosci. 24 (2001), 1121-1159 in which Table 1catalogs the unique members of tauopathies or Sergeant et al., Bioch.Biophy. Acta 1739 (2005), 179-97, with a list in FIG. 2 therein.

In this specification, the terms “tau”, is used interchangeable tospecifically refer to the native monomer form of tau. The term “tau” isalso used to generally identify other conformers of tau, for example,oligomers or aggregates of tau. The term “tau” is also used to refercollectively to all types and forms of tau. Due to alternative splicing6 tau isoforms are present in the human brain. The protein sequences forthese isoforms are:

Isoform Fetal-tau of 352aa (SEQ ID NO: 1)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQ GLIsoform Tau-B of 381aa (SEQ ID NO: 2)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL Isoform Tau-C of 410aa (SEQ ID NO: 3)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADE VSASLAKQGLIsoform Tau-D of 383aa (SEQ ID NO: 4)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL Isoform Tau-E of 412aa (SEQ ID NO: 5)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLA DEVSASLAKQGLIsoform Tau-F of 441aa (SEQ ID NO: 6)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

The “wild type” tau amino acid sequence is represented by isoform Tau-Fof 441aa (SEQ ID NO:6) further also referenced to as “hTau40”, “TauF”,“Tau-4” or “full-length tau”. The amino acid sequence of tau can beretrieved from the literature and pertinent databases; see Goedert etal., Proc. Natl. Acad. Sci. USA 85 (1988), 4051-4055, Goedert et al.,EMBO J. 8(1989), 393-399, Goedert et al., EMBO J. 9 (1990), 4225-4230and GenBank UniProtKB/swissprot: locus TAU_HUMAN, accession numbersP10636-2 (Fetal-tau) and P10636-4 to -8 (Isoforms B to F).

Another striking feature of tau protein is phosphorylation, which occursat about 30 of 79 potential serine (Ser) and threonine (Thr)phosphorylation sites. Tau is highly phosphorylated during the braindevelopment. The degree of phosphorylation declines in adulthood. Someof the phosphorylation sites are located within the microtubule bindingdomains of tau, and it has been shown that an increase of tauphosphorylation negatively regulates the binding of microtubules. Forexample, Ser262 and Ser396, which lie within or adjacent to microtubulebinding motifs, are hyperphosphorylated in the tau proteins of theabnormal paired helical filaments (PHFs)), a major component of theneurofibrillary tangles (NFTs) in the brain of AD patients. PHFs arefilamentous aggregates of tau proteins which are abnormallyhyperphosphorylated and can be stained with specific anti-tau antibodiesand detected by light microscopy. The same holds true for so calledstraight tau filaments. PHFs form twisted ribbons consisting of twofilaments twisted around one another with a periodicity of about 80 nm.These pathological features are commonly referred to as “tau-pathology”,“tauopathology” or “tau-related pathology”. For a more detaileddescription of neuropathological features of tauopathies refer to Lee etal., Annu. Rev. Neurosci. 24 (2001), 1121-1159 and Götz, Brain. Res.Rev. 35 (2001), 266-286, the disclosure content of which is incorporatedherein by reference. Physiological tau protein stabilizes microtubulesin neurons. Pathological phyosphorylation leads to abnormal taulocalization and aggregation, which causes destabilization ofmicrotubules and impaired cellular transport. Aggregated tau isneurotoxic in vitro (Khlistunova et al., J. Biol. Chem. 281 (2006),1205-1214). The exact neurotoxic species remains unclear, however, as dothe mechanism(s) by which they lead to neuronal death. Aggregates of taucan be observed as the main component of neurofibrillary tangles (NFT)in many tauopathies, such as Alzheimer's disease (AD), Frontotemporaldementias, supranuclear palsy, Pick's disease, Argyrophilic graindisease (AGD), corticobasal degeneration, FTDP-17, Parkinson's disease,Dementia pugilistica (Reviewed in Gendron and Petrucelli, Mol.Neurodegener. 4:13 (2009)). Besides these observations, evidence emergesthat tau-mediated neuronal death can occur even in the absence of tangleformation. Soluble phospho-tau species are present in CSF (Aluise etal., Biochim. Biophys. Acta. 1782 (2008), 549-558). Tau aggregates cantransmit a misfolded state from the outside to the inside of a cell andtransfer between co-cultured cells (Frost et al., J. Biol. Chem. 284(2009), 12845-12852).

In addition to the involvement in neurodegenerative tauopathies,observed alterations in tau phosphorylation during and afterischemia/reperfusion suggest tau playing a crucial role in neuronaldamage and clinical pathophysiology of neurovascular disorders such asischemic stroke (Zheng et al., J. Cell. Biochem. 109 (2010), 26-29).

The human anti-tau antibodies disclosed herein specifically bind tau andepitopes thereof and to various conformations of tau and epitopesthereof. For example, disclosed herein are antibodies that specificallybind tau, tau in its full-length, pathologically modified tau isoformsand tau aggregates. As used herein, reference to an antibody that“specifically binds”, “selectively binds”, or “preferentially binds” taurefers to an antibody that does not bind other unrelated proteins. Inone example, a tau antibody disclosed herein can bind tau or an epitopethereof and show no binding above about 1.5 times background for otherproteins. An antibody that “specifically binds” or “selectively binds” atau conformer refers to an antibody that does not bind all conformationsof tau, i.e., does not bind at least one other tau conformer. Forexample, disclosed herein are antibodies that can preferentially bind toaggregated forms of tau in AD tissue. Since the human anti-tauantibodies of the present invention have been isolated from a pool ofhealthy human subjects exhibiting an tau-specific immune response thetau antibodies of the present invention may also be called “humanauto-antibodies” in order to emphasize that those antibodies were indeedexpressed by the subjects and have not been isolated from, for example ahuman immunoglobulin expressing phage library, which hithertorepresented one common method for trying to provide human-likeantibodies.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms.

The term “polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to antibodies or antibody polypeptides of thepresent invention include any polypeptides which retain at least some ofthe antigen-binding properties of the corresponding native bindingmolecule, antibody, or polypeptide. Fragments of polypeptides of thepresent invention include proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of antibodies and antibody polypeptides ofthe present invention include fragments as described above, and alsopolypeptides with altered amino acid sequences due to amino acidsubstitutions, deletions, or insertions. Variants may occur naturally orbe non-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of tau specific binding molecules,e.g., antibodies and antibody polypeptides of the present invention, arepolypeptides which have been altered so as to exhibit additionalfeatures not found on the native polypeptide. Examples include fusionproteins. Variant polypeptides may also be referred to herein as“polypeptide analogs”. As used herein a “derivative” of a bindingmolecule or fragment thereof, an antibody, or an antibody polypeptiderefers to a subject polypeptide having one or more residues chemicallyderivatized by reaction of a functional side group. Also included as“derivatives” are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexample, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding abinding molecule, an antibody, or fragment, variant, or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” or “operablylinked” if induction of promoter function results in the transcriptionof mRNA encoding the desired gene product and if the nature of thelinkage between the two DNA fragments does not interfere with theability of the expression regulatory sequences to direct the expressionof the gene product or interfere with the ability of the DNA template tobe transcribed. Thus, a promoter region would be operably associatedwith a nucleic acid encoding a polypeptide if the promoter was capableof effecting transcription of that nucleic acid. The promoter may be acell-specific promoter that directs substantial transcription of the DNAonly in predetermined cells. Other transcription control elements,besides a promoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full-length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

Unless stated otherwise, the terms “disorder” and “disease” are usedinterchangeably herein.

A “binding molecule” as used in the context of the present inventionrelates primarily to antibodies, and fragments thereof, but may alsorefer to other non-antibody molecules that bind to tau including but notlimited to hormones, receptors, ligands, major histocompatibilitycomplex (MHC) molecules, chaperones such as heat shock proteins (HSPs)as well as cell-cell adhesion molecules such as members of the cadherin,intergrin, C-type lectin and immunoglobulin (Ig) superfamilies. Thus,for the sake of clarity only and without restricting the scope of thepresent invention most of the following embodiments are discussed withrespect to antibodies and antibody-like molecules which represent aspecific embodiment of binding molecules for the development oftherapeutic and diagnostic agents.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin is a tau-binding molecule whichcomprises at least the variable domain of a heavy chain, and normallycomprises at least the variable domains of a heavy chain and a lightchain. Basic immunoglobulin structures in vertebrate systems arerelatively well understood; see, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theIgG class of immunoglobulin molecules. With regard to IgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the V_(L) domain and V_(H) domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the V_(H) and V_(L) chains. Anyantibody or immunoglobulin fragment which contains sufficient structureto specifically bind to tau is denoted herein interchangeably as a“binding fragment” or an “immunospecific fragment.”

In naturally occurring antibodies, an antibody comprises sixhypervariable regions, sometimes called “complementarity determiningregions” or “CDRs” present in each antigen-binding domain, which areshort, non-contiguous sequences of amino acids that are specificallypositioned to form the antigen-binding domain as the antibody assumesits three dimensional configuration in an aqueous environment. The“CDRs” are flanked by four relatively conserved “framework” regions or“FRs” which show less inter-molecular variability. The framework regionslargely adopt a β-sheet conformation and the CDRs form loops whichconnect, and in some cases form part of, the β-sheet structure. Thus,framework regions act to form a scaffold that provides for positioningthe CDRs in correct orientation by inter-chain, non-covalentinteractions. The antigen-binding domain formed by the positioned CDRsdefines a surface complementary to the epitope on the immunoreactiveantigen. This complementary surface promotes the non-covalent binding ofthe antibody to its cognate epitope. The amino acids comprising the CDRsand the framework regions, respectively, can be readily identified forany given heavy or light chain variable region by one of ordinary skillin the art, since they have been precisely defined; see, “Sequences ofProteins of Immunological Interest,” Kabat, E., et al., U.S. Departmentof Health and Human Services, (1983); and Chothia and Lesk, J. Mol.Biol., 196 (1987), 901-917, which are incorporated herein by referencein their entireties.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaand Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporatedherein by reference, where the definitions include overlapping orsubsets of amino acid residues when compared against each other.Nevertheless, application of either definition to refer to a CDR of anantibody or variants thereof is intended to be within the scope of theterm as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 1 as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular hypervariable region orCDR of the human IgG subtype of antibody given the variable region aminoacid sequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,variant, or derivative thereof of the present invention are according tothe Kabat numbering system, which however is theoretical and may notequally apply every antibody of the present invention. For example,depending on the position of the first CDR the following CDRs might beshifted in either direction.

Antibodies or antigen-binding fragments, immunospecific fragments,variants, or derivatives thereof of the invention include, but are notlimited to, polyclonal, monoclonal, multispecific, human, humanized,primatized, murinized or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)₂, Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a V_(L) or V_(H) domain, fragmentsproduced by a Fab expression library, and anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies disclosedherein). ScFv molecules are known in the art and are described, e.g., inU.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

In one embodiment, the antibody of the present invention is not IgM or aderivative thereof with a pentavalent structure. Particular, in specificapplications of the present invention, especially therapeutic use, IgMsare less useful than IgG and other bivalent antibodies or correspondingbinding molecules since IgMs due to their pentavalent structure and lackof affinity maturation often show unspecific cross-reactivities and verylow affinity.

In a particular embodiment, the antibody of the present invention is nota polyclonal antibody, i.e. it substantially consists of one particularantibody species rather than being a mixture obtained from a plasmaimmunoglobulin sample.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded in the invention are tau-binding fragments also comprising anycombination of variable region(s) with a hinge region, CH1, CH2, and CH3domains. Antibodies or immunospecific fragments thereof of the presentinvention may be from any animal origin including birds and mammals. Inone embodiment, the antibodies are human, murine, donkey, rabbit, goat,guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be condricthoid in origin (e.g.,from sharks).

In one aspect, the antibody of the present invention is a humanmonoclonal antibody isolated from a human. Optionally, the frameworkregion of the human antibody is aligned and adopted in accordance withthe pertinent human germ line variable region sequences in the database;see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRCCentre for Protein Engineering (Cambridge, UK). For example, amino acidsconsidered to potentially deviate from the true germ line sequence couldbe due to the PCR primer sequences incorporated during the cloningprocess. Compared to artificially generated human-like antibodies suchas single chain antibody fragments (scFvs) from a phage displayedantibody library or xenogeneic mice the human monoclonal antibody of thepresent invention is characterized by (i) being obtained using the humanimmune response rather than that of animal surrogates, i.e. the antibodyhas been generated in response to natural tau in its relevantconformation in the human body, (ii) having protected the individual oris at least significant for the presence of tau, and (iii) since theantibody is of human origin the risks of cross-reactivity againstself-antigens is minimized Thus, in accordance with the presentinvention the terms “human monoclonal antibody”, “human monoclonalautoantibody”, “human antibody” and the like are used to denote a taubinding molecule which is of human origin, i.e. which has been isolatedfrom a human cell such as a B cell or hybridoma thereof or the cDNA ofwhich has been directly cloned from mRNA of a human cell, for example ahuman memory B cell. A human antibody is still “human” even if aminoacid substitutions are made in the antibody, e.g., to improve bindingcharacteristics.

Antibodies derived from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al., are denoted human-likeantibodies in order distinguish them from truly human antibodies of thepresent invention.

For example, the paring of heavy and light chains of human-likeantibodies such as synthetic and semi-synthetic antibodies typicallyisolated from phage display do not necessarily reflect the originalparing as it occurred in the original human B cell. Accordingly Fab andscFv fragments obtained from recombinant expression libraries ascommonly used in the prior art can be considered as being artificialwith all possible associated effects on immunogenicity and stability.

In contrast, the present invention provides isolated affinity-maturedantibodies from selected human subjects, which are characterized bytheir therapeutic utility and their tolerance in man.

As used herein, the term “murinized antibody” or “murinizedimmunoglobulin” refers to an antibody comprising one or more CDRs from ahuman antibody of the present invention; and a human framework regionthat contains amino acid substitutions and/or deletions and/orinsertions that are based on a mouse antibody sequence. The humanimmunoglobulin providing the CDRs is called the “parent” or “acceptor”and the mouse antibody providing the framework changes is called the“donor”. Constant regions need not be present, but if they are, they areusually substantially identical to mouse antibody constant regions, i.e.at least about 85-90%, about 95%, about 96%, about 97%, about 98%, about99% or more identical. Hence, in some embodiments, a full-lengthmurinized human heavy or light chain immunoglobulin contains a mouseconstant region, human CDRs, and a substantially human framework thathas a number of “murinizing” amino acid substitutions. Typically, a“murinized antibody” is an antibody comprising a murinized variablelight chain and/or a murinized variable heavy chain. For example, amurinized antibody would not encompass a typical chimeric antibody,e.g., because the entire variable region of a chimeric antibody isnon-mouse. A modified antibody that has been “murinized” by the processof “murinization” binds to the same antigen as the parent antibody thatprovides the CDRs and is usually less immunogenic in mice, as comparedto the parent antibody.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody or diabody.

In another embodiment, the antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein are composed of asingle polypeptide chain such as scFvs and are to be expressedintracellularly (intrabodies) for potential in vivo therapeutic anddiagnostic applications.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a CH1 domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. In one embodiment,the light chain portion comprises at least one of a V_(L) or CL domain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes may contain at least seven, at least nine or between at leastabout 15 to about 30 amino acids. Since a CDR can recognize an antigenicpeptide or polypeptide in its tertiary form, the amino acids comprisingan epitope need not be contiguous, and in some cases, may not even be onthe same peptide chain. In the present invention, a peptide orpolypeptide epitope recognized by antibodies of the present inventioncontains a sequence of at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 15, at least 20, at least25, or between about 5 to about 30, about 10 to about 30 or about 15 toabout 30 contiguous or non-contiguous amino acids of tau.

By “specifically binding”, or “specifically recognizing”, usedinterchangeably herein, it is generally meant that a binding molecule,e.g., an antibody binds to an epitope via its antigen-binding domain,and that the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. A skilled artisan understands thatan antibody may specifically bind to, or specifically recognize anisolated polypeptide comprising, or consisting of, amino acid residuescorresponding to a linear portion of a non-contiguous epitope. The term“specificity” is used herein to qualify the relative affinity by which acertain antibody binds to a certain epitope. For example, antibody “A”may be deemed to have a higher specificity for a given epitope thanantibody “B,” or antibody “A” may be said to bind to epitope “C” with ahigher specificity than it has for related epitope “D”.

Where present, the term “immunological binding characteristics,” orother binding characteristics of an antibody with an antigen, in all ofits grammatical forms, refers to the specificity, affinity,cross-reactivity, and other binding characteristics of an antibody.

By “preferentially binding”, it is meant that the binding molecule,e.g., antibody specifically binds to an epitope more readily than itwould bind to a related, similar, homologous, or analogous epitope.Thus, an antibody which “preferentially binds” to a given epitope wouldmore likely bind to that epitope than to a related epitope, even thoughsuch an antibody may cross-react with the related epitope.

By way of non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it bindssaid first epitope with a dissociation constant (K_(D)) that is lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstantigen preferentially if it binds the first epitope with an affinitythat is at least one order of magnitude less than the antibody's K_(D)for the second epitope. In another non-limiting example, an antibody maybe considered to bind a first epitope preferentially if it binds thefirst epitope with an affinity that is at least two orders of magnitudeless than the antibody's K_(D) for the second epitope.

In another non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it binds thefirst epitope with an off rate (k(off)) that is less than the antibody'sk(off) for the second epitope. In another non-limiting example, anantibody may be considered to bind a first epitope preferentially if itbinds the first epitope with an affinity that is at least one order ofmagnitude less than the antibody's k(off) for the second epitope. Inanother non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with anaffinity that is at least two orders of magnitude less than theantibody's k(off) for the second epitope.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind a tau or afragment or variant thereof with an off rate (k(off)) of less than orequal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹. In oneembodiment, an antibody of the invention may be said to bind tau or afragment or variant thereof with an off rate (k(off)) less than or equalto 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹,10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind tau or afragment or variant thereof with an on rate (k(on)) of greater than orequal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹sec⁻¹. In one embodiment, an antibody of the invention may be said tobind tau or a fragment or variant thereof with an on rate (k(on))greater than or equal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹,or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

A binding molecule, e.g., an antibody is said to competitively inhibitbinding of a reference antibody to a given epitope if it preferentiallybinds to that epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitionmay be determined by any method known in the art, for example,competition ELISA assays. An antibody may be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%. Askilled artisan understands that the binding of an antibody to itsepitope may also be competitively inhibited by a binding molecule thatis not an antibody. For example, the specific binding of an antibodydescribed herein to tau, e.g., hTau40, may be competitively inhibited bymicrotubules.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of a bindingmolecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed. (1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population ofimmunoglobulins and an antigen, that is, the functional combiningstrength of an immunoglobulin mixture with the antigen; see, e.g.,Harlow at pages 29-34. Avidity is related to both the affinity ofindividual immunoglobulin molecules in the population with specificepitopes, and also the valencies of the immunoglobulins and the antigen.For example, the interaction between a bivalent monoclonal antibody andan antigen with a highly repeating epitope structure, such as a polymer,would be one of high avidity. The affinity or avidity of an antibody foran antigen can be determined experimentally using any suitable method;see, for example, Berzofsky et al., “Antibody-Antigen Interactions” InFundamental Immunology, Paul, W. E., Ed., Raven Press New York, N.Y.(1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N.Y.(1992), and methods described herein. General techniques for measuringthe affinity of an antibody for an antigen include ELISA, RIA, andsurface plasmon resonance. The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions, e.g., salt concentration, pH. Thus, measurements of affinityand other antigen-binding parameters, e.g., K_(D), IC₅₀, are preferablymade with standardized solutions of antibody and antigen, and astandardized buffer.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their cross-reactivity. As used herein, theterm “cross-reactivity” refers to the ability of an antibody, specificfor one antigen, to react with a second antigen; a measure ofrelatedness between two different antigenic substances. Thus, anantibody is cross reactive if it binds to an epitope other than the onethat induced its formation. The cross reactive epitope generallycontains many of the same complementary structural features as theinducing epitope, and in some cases, may actually fit better than theoriginal.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their binding affinity to tau. In oneembodiment, binding affinities include those with a dissociationconstant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M,10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M,10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M,or 10⁻¹⁵ M.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “V_(H) domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the V_(H) domain and is amino terminal to the hinge regionof an immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat EA et al. op. cit). The CH2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains; see Roux et al., J.Immunol. 161 (1998), 4083.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the terms “linked”, “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of such mRNA intopolypeptide(s). If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., a messenger RNA produced bytranscription of a gene, or a polypeptide which is translated from atranscript. Gene products described herein further include nucleic acidswith post transcriptional modifications, e.g., polyadenylation, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, proteolytic cleavage, and the like.

As used herein, the term “sample” refers to any biological materialobtained from a subject or patient. In one aspect, a sample can compriseblood, cerebrospinal fluid (“CSF”), or urine. In other aspects, a samplecan comprise whole blood, plasma, B cells enriched from blood samples,and cultured cells (e.g., B cells from a subject). A sample can alsoinclude a biopsy or tissue sample including neural tissue. In stillother aspects, a sample can comprise whole cells and/or a lysate of thecells. Blood samples can be collected by methods known in the art. Inone aspect, the pellet can be resuspended by vortexing at 4° C. in 200μl buffer (20 mM Tris, pH. 7.5, 0.5% Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1MNaCl, IX Sigma Protease Inhibitor, and IX Sigma Phosphatase Inhibitors 1and 2). The suspension can be kept on ice for 20 minutes withintermittent vortexing. After spinning at 15,000×g for 5 minutes atabout 4° C., aliquots of supernatant can be stored at about −70° C.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development ofParkinsonism. Beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the manifestation of the condition or disorder is to beprevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

II. Antibodies

The present invention generally relates to human anti-tau antibodies andantigen-binding fragments thereof. In one embodiment, an antibody of thepresent invention demonstrates the immunological binding characteristicsand/or biological properties as outlined for the antibodies illustratedin the Examples. In accordance with the present invention humanmonoclonal antibodies specific for tau were cloned from a pool ofhealthy human subjects.

In the course of the experiments performed in accordance with thepresent invention initial attempts failed to clone tau specificantibodies but almost always resulted in false-positive clones. In orderto circumvent this problem, antibodies in conditioned media of humanmemory B cell cultures were screened in parallel for binding torecombinant tau protein, PHFTau extracted from AD brain, healthy controlbrain extracts and bovine serum albumin (BSA). Only B-cell cultures thatwere positive for recombinant tau and/or PHFTau but not control brainextract or BSA were subjected to antibody cloning.

Initial attempts to isolating to specific antibodies were focused atpools of healthy human subjects with high plasma binding activity totau, suggestive of elevated levels of circulating tau antibodies plasma.Unexpectedly, these attempts failed to produce tau specific human memoryB cells and the antibodies described in the current invention wereisolated from pools of healthy human subjects that were not preselectedfor high tau plasma reactivity or had low plasma reactivity to tau.

Due to this measure, several antibodies could be isolated. Selectedantibodies were further analyzed for class and light chain subclassdetermination. Selected relevant antibody messages from memory B cellcultures are then transcribed by RT-PCR, cloned and combined intoexpression vectors for recombinant production; see the appendedExamples. Recombinant expression of the human antibodies in HEK293 orCHO cells and the subsequent characterization of their bindingspecificities towards full-length tau (FIG. 2, FIG. 7 and FIG. 12),pathologically modified forms thereof on Western Blot (FIG. 3 and FIG.8) and their distinctive binding to pathologically aggregated tauconfirmed that for the first time human antibodies have been cloned thatare highly specific for tau and recognize distinctive the pathologicallymodified forms of tau protein.

Thus, the present invention generally relates to an isolated naturallyoccurring human monoclonal anti-tau antibody and binding fragments,derivatives and variants thereof. In one embodiment of the invention,the antibody is capable of specifically binding full-length recombinanttau and/or the pathologically aggregated and/or phosphorylated form(PHFTau) isolated from AD brain under denaturing conditions on WesternBlot; see FIG. 3 and FIG. 8.

In one embodiment, the present invention is directed to an anti-tauantibody, or antigen-binding fragment, variant or derivatives thereof,where the antibody specifically binds to the same epitope of tau as areference antibody selected from the group consisting of NI-105-4E4,NI-105-24B2 or NI-105.4A3. In addition, preliminary results of directELISA assays performed with the exemplary antibody NI-105-4E4 revealedthat NI-105-4E4 specifically recognizes the C-terminus of tau.Additional assays performed suggest that NI-105.4E4 recognizes adiscontinuous epitope comprising two linear sequences: a first linearsequence within the R4 microtubule binding domain and a second linearsequence within the region between the R4 and C domains as depicted inFIG. 11. In one embodiment, a linear polypeptide comprised by anon-continuous epitope, or an epitope recognized by an antibody providedby this invention is located in the microtubule binding domain of tau,which is masked in physiological microtubule-associated tau. Epitopemapping identified a first sequence within the microtubule bindingdomain of human tau including aa337-343 VEVKSEK (SEQ ID NO:7) as aunique linear polypeptide comprised by the epitope recognized byantibody NI-105.4E4 of this invention. Additional experiments andcomparison with a commercially available AT180 mouse monoclonal tauantibody confirmed that NI-105-4E4 specifically recognizes the uniqueepitope of SEQ ID NO:7. Most advantageously, the SEQ ID NO:7 epitoperecognized by the antibody NI-105.4E4 of this invention is 100%conserved in all 6 tau isoforms present in the human brain of the aminoacid sequences represented by SEQ ID NO:1 to 6 and in other species,such as mouse and rat as well providing an additional research tool inrespective animal models with the antibodies of the present invention.Further experimentation showed that residues 3 and 6 of the SEQ ID NO: 7polypeptide, corresponding to residues V339 and E342 of SEQ ID NO: 6,contribute to the binding of NI-105.4E4. Epitope mapping furtheridentified a second sequence (SEQ ID NO:41) within the microtubulebinding domain of human tau including aa387-397 of SEQ ID NO:6 as aunique linear polypeptide comprised by the epitope recognized byantibody NI-105.4E4 of this invention. Residues 1, 5 and 9 of SEQ ID NO:41, corresponding to residues D387, E391 and K395 of SEQ ID NO: 6,contribute to the binding of NI-105.4E4.

In one embodiment, an antibody described herein specifically binds totau at an epitope comprising the amino acid residues of SEQ ID NO: 7. Inanother embodiment, an antibody described herein specifically binds totau at an epitope comprising the amino acid residues of SEQ ID: 41. In aspecific embodiment, an antibody described herein specifically binds totau at an epitope comprising the amino acid residues of SEQ ID NO:7 andSEQ ID NO:41. In a further embodiment, an antibody described hereinspecifically binds to tau at an epitope comprising one or more aminoacid residues selected from the group consisting of residues V339, E342,D387, E391 and K395 of SEQ ID NO:6. The epitope may comprise any one,any two, any three, any four or all five residues from the groupconsisting of residues V339, E342, D387, E391 and K395 of SEQ ID NO:6.In a specific embodiment, tau is hTau40.

In one embodiment, an antibody described herein binds to tau at anepitope comprising the microtubule binding domain of tau. In a specificembodiment, an antibody described herein binds to tau at an epitopecomprising amino acid residues from the R4 region of tau as depicted inFIG. 11. In one embodiment, an antibody described herein competes withmicrotubules for specific binding to tau. In another embodiment, anantibody described herein has reduced binding affinity to microtubuleassociated tau compared to the antibodies binding affinity to tau noassociated with microtubules. In a further embodiment, an antibodydescribed herein does not bind, or substantially does not bind to tauassociated with microtubules. In specific embodiments, the tau proteinmay be native tau protein or recombinant tau protein. In a specificembodiment, tau is hTau40.

Epitope mapping further identified a sequence (SEQ ID NO:42) of humantau including aa35-49 of SEQ ID NO:6 as a unique linear epitoperecognized by antibody NI-105.4A3 of this invention. Residues 6, 7 and10 of SEQ ID NO: 42, corresponding to residues D40, A41 and K44 of SEQID NO: 6, contribute to the binding of NI-105.4A3. In one embodiment, anantibody described herein specifically binds to tau at an epitopecomprising the amino acid residues of SEQ ID NO: 42. In a furtherembodiment, an antibody described herein specifically binds to tau at anepitope comprising one or more amino acid residues selected from thegroup consisting of residues D40, A41 and K44 of SEQ ID NO:6. Theepitope may comprise any one, any two, or all any three residues fromthe group consisting of residues D40, A41 and K44 of SEQ ID NO:6. In aspecific embodiment, tau is hTau40.

Further, without intending to be bound by initial experimentalobservations as demonstrated in the Examples and shown in FIG. 6, thehuman monoclonal NI-105-4E4 anti-tau antibody of the present inventionis characterized in specifically binding pathologically aggregated tauand not substantially recognizing tau in the physiological form in braintissue. In one embodiment, a human anti-tau antibody of the presentinvention may specifically bind pathologically aggregated tau and notsubstantially bind tau in the physiological form in brain tissue. Inaddition, a human anti-tau antibody of the present invention may befurther characterized by its ability to recognize tau at the pre-tanglestage, in neurofibrillary tangles (NFT), neutropil threads and/ordystrophic neurites in the brain. Hence, the present invention providesa set of human tau antibodies with binding specificities, which are thusparticularly useful for diagnostic and therapeutic purposes.

In one embodiment, the antibody of the present invention exhibits thebinding properties of the exemplary NI-105-4E4 antibody as described inthe Examples. In addition, or alternatively, an anti-tau antibody of thepresent invention preferentially recognizes pathologically aggregatedtau rather than physiological forms, in particular when analyzedaccording to Example 4. In addition, or alternatively, an anti-tauantibody of the present invention binds to disease causing mutants ofhuman tau, in particular those described in Example 4. In this context,the binding specificities may be in the range as shown for the exemplaryNI-105.4E4, NI-1054A3 and NI-105.24B2 antibodies in FIG. 2, FIG. 12 andFIG. 7, respectively, i.e. having half maximal effective concentrations(EC50) of about 100 pM to 100 nM, or an EC50 of about 100 pM to 10 nMfor wild-type tau.

Hence, an anti-tau antibody of the present invention bindspreferentially to pathological modified forms of tau in brain, e.g.pathological aggregates of tau as exemplified by immunohistochemicalstaining described in Example 4. In another embodiment an anti-tauantibody of the present invention preferentially binds to bothrecombinant tau and pathologically modified forms of tau as exemplifiedin Example 2 by Western Blot.

The present invention is also drawn to an antibody, or antigen-bindingfragment, variant or derivatives thereof, where the antibody comprisesan antigen-binding domain identical to that of an antibody selected fromthe group consisting of NI-105-4E4, NI-105-24B2 and NI-105.4A3.

The present invention further exemplifies several such bindingmolecules, e.g.

antibodies and binding fragments thereof, which may be characterized bycomprising in their variable region, e.g. binding domain at least onecomplementarity determining region (CDR) of the VH and/or VL variableregion comprising any one of the amino acid sequences depicted inFIG. 1. The corresponding nucleotide sequences encoding theabove-identified variable regions are set forth in Table 2 below. Anexemplary set of CDRs of the above amino acid sequences of the VH and/orVL region as depicted in FIG. 1. However, as discussed in the followingthe person skilled in the art is well aware of the fact that in additionor alternatively CDRs may be used, which differ in their amino acidsequence from those set forth in FIG. 1 by one, two, three or even moreamino acids in case of CDR2 and CDR3.

In one embodiment, an antibody of the present invention comprises atleast one CDR comprising, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO: 23-25, 26-28, 29-31,32-34, 35-37 and 38-40. In one embodiment, an antibody of the presentinvention comprises one, two, three, four, five or six CDRs comprising,or consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 23-25, 26-28, 29-31, 32-34, 35-37 and 38-40.The antibody may comprise a heavy chain variable region comprising a VHCDR1 of SEQ ID NO: 23, 29 or 35; a VH CDR2 of SEQ ID NO: 24,30 or 36; ora VH CDR3 of SEQ ID NO: 25, 31 or 37. The antibody may comprise a lightchain variable region comprising a VL CDR1 of SEQ ID NO: 26, 32 or 38; aVL CDR2 of SEQ ID NO: 27, 33 or 39; or a VL CDR3 of SEQ ID NO: 28, 34 or40. The antibody may comprise a heavy chain variable region comprising aVH CDR1 of SEQ ID NO: 23, 29 or 35; a VH CDR2 of SEQ ID NO: 24,30 or 36;or a VH CDR3 of SEQ ID NO: 25, 31 or 37, and may further comprise alight chain variable region comprising a VL CDR1 of SEQ ID NO: 26, 32 or38; a VL CDR2 of SEQ ID NO: 27, 33 or 39; or a VL CDR3 of SEQ ID NO: 28,34 or 40.

In one embodiment, an antibody of the present invention may comprise aheavy chain variable region comprising a VH CDR1 of SEQ ID NO: 23, 29 or35; a VH CDR2 of SEQ ID NO: 24,30 or 36; and a VH CDR3 of SEQ ID NO: 25,31 or 37. In one embodiment, an antibody of the present invention maycomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO: 26, 32 or 38; a VL CDR2 of SEQ ID NO: 27, 33 or 39; and a VL CDR3 ofSEQ ID NO: 28, 34 or 40. The antibody may further comprise a heavy chainvariable region comprising a VH CDR1 of SEQ ID NO: 23, 29 or 35; a VHCDR2 of SEQ ID NO: 24,30 or 36; and a VH CDR3 of SEQ ID NO: 25, 31 or37, and may further comprise a light chain variable region comprising aVL CDR1 of SEQ ID NO: 26, 32 or 38; a VL CDR2 of SEQ ID NO: 27, 33 or39; and a VL CDR3 of SEQ ID NO: 28, 34 or 40.

In one embodiment, an antibody of the present invention may comprise aheavy chain variable region comprising a VH CDR1 of SEQ ID NO: 23; a VHCDR2 of SEQ ID NO: 24; and a VH CDR3 of SEQ ID NO: 25. In oneembodiment, an antibody of the present invention may comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 29; a VH CDR2of SEQ ID NO: 30; and a VH CDR3 of SEQ ID NO: 31. In one embodiment, anantibody of the present invention may comprise a heavy chain variableregion comprising a VH CDR1 of SEQ ID NO: 35; a VH CDR2 of SEQ ID NO:36; and a VH CDR3 of SEQ ID NO: 37.

In one embodiment, an antibody of the present invention may comprise alight chain variable region comprising a VL CDR1 of SEQ ID NO: 26; a VLCDR2 of SEQ ID NO: 27; and a VL CDR3 of SEQ ID NO: 28. In oneembodiment, an antibody of the present invention may comprise a lightchain variable region comprising a VL CDR1 of SEQ ID NO: 32; a VL CDR2of SEQ ID NO: 33; and a VL CDR3 of SEQ ID NO: 34. In one embodiment, anantibody of the present invention may comprise a light chain variableregion comprising a VL CDR1 of SEQ ID NO: 38; a VL CDR2 of SEQ ID NO:39; and a VL CDR3 of SEQ ID NO: 40.

In one embodiment, an antibody of the present invention may comprise aheavy chain variable region comprising a VH CDR1 of SEQ ID NO: 23; a VHCDR2 of SEQ ID NO: 24; and a VH CDR3 of SEQ ID NO: 25, and may furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO: 26; a VL CDR2 of SEQ ID NO: 27; and a VL CDR3 of SEQ ID NO: 28.

In one embodiment, an antibody of the present invention may comprise aheavy chain variable region comprising a VH CDR1 of SEQ ID NO: 29; a VHCDR2 of SEQ ID NO: 30; and a VH CDR3 of SEQ ID NO: 31 and may furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO: 32; a VL CDR2 of SEQ ID NO: 33; and a VL CDR3 of SEQ ID NO: 34.

In one embodiment, an antibody of the present invention may comprise aheavy chain variable region comprising a VH CDR1 of SEQ ID NO: 35; a VHCDR2 of SEQ ID NO: 36; and a VH CDR3 of SEQ ID NO: 37 and may furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO: 38; a VL CDR2 of SEQ ID NO: 39; and a VL CDR3 of SEQ ID NO: 40.

In one embodiment, the antibody of the present invention is any one ofthe antibodies comprising an amino acid sequence of the VH and/or VLregion as depicted in FIG. 1. In one embodiment, the antibody of thepresent invention is characterized by the preservation of the cognatepairing of the heavy and light chain as was present in the human B-cell.

In one embodiment, an antibody of the present invention comprises aheavy chain variable region (VH) comprising, or consisting of an aminoacid sequence selected from the group consisting of SEQ ID NO: 9, 13, 17and 93. In one embodiment, an antibody of the present inventioncomprises a light chain variable region (VL) comprising, or consistingof an amino acid sequence selected from the group consisting of SEQ IDNO: 11, 15 and 19. In one embodiment, an antibody of the presentinvention comprises a heavy chain variable region (VH) comprising, orconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 9, 13, 17 and 93, and further comprises a light chainvariable region (VL) comprising, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO: 11, 15 and 19. In aspecific embodiment, the antibody comprises a VH of SEQ ID NO: 9 and aVL of SEQ ID NO: 11; or a VH of SEQ ID NO: 93 and a VL of SEQ ID NO: 11;a VH of SEQ ID NO: 13 and a VL of SEQ ID NO: 15; or a VH of SEQ ID NO:17 and a VL of SEQ ID NO: 19.

Alternatively, the antibody of the present invention is an antibody orantigen-binding fragment, derivative or variant thereof, which competesfor binding to tau, such as, for example, hTau40, with at least one ofthe antibodies having the VH and/or VL region as depicted in FIG. 1. Inone embodiment, an antibody of the present invention competes forspecific binding to hTau40 with NI-105-4E4, NI-105-24B2 or NI-105.4A3.Those antibodies may be human as well, in particular for therapeuticapplications. Alternatively, the antibody is a murine, murinized andchimeric murine-human antibody, which are particularly useful fordiagnostic methods and studies in animals.

In one embodiment the antibody of the present invention is provided bycultures of single or oligoclonal B-cells that are cultured and thesupernatant of the culture, which contains antibodies produced by saidB-cells is screened for presence and affinity of anti-tau antibodiestherein. The screening process comprises the steps of a sensitive tissueamyloid plaque immunoreactivity (TAPIR) assay such as described ininternational application WO2004/095031, the disclosure content of whichis incorporated herein by reference; screen on brain sections forbinding to PHFTau; screening for binding of a peptide derived from tauof the amino acid sequence represented by SEQ ID NO:6 with phosphategroups on amino acids Ser-202 and Thr-205; on amino acid Thr-231; and/oron amino acids Ser-396 and Ser-404 of said sequence; a screen forbinding of recombinant human tau of the amino acid sequence representedby SEQ ID NO:6 and isolating the antibody for which binding is detectedor the cell producing said antibody.

As mentioned above, due to its generation upon a human immune responsethe human monoclonal antibody of the present invention will recognizeepitopes which are of particular pathological relevance and which mightnot be accessible or less immunogenic in case of immunization processesfor the generation of, for example, mouse monoclonal antibodies and invitro screening of phage display libraries, respectively. Accordingly,it is prudent to stipulate that the epitope of the human anti-tauantibody of the present invention is unique and no other antibody whichis capable of binding to the epitope recognized by the human monoclonalantibody of the present invention exists; see also FIG. 11 which showsthe unique epitope of antibodies NI-105.4E4 and NI-105.4A3. Therefore,the present invention also extends generally to anti-tau antibodies andtau binding molecules which compete with the human monoclonal antibodyof the present invention for specific binding to tau. The presentinvention is more specifically directed to an antibody, orantigen-binding fragment, variant or derivatives thereof, where theantibody specifically binds to the same epitope of tau as a referenceantibody selected from the group consisting of NI-105.4E4, NI-105.24B2and NI-105.4A3.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as tau. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay; see Stahli et al.,Methods in Enzymology 9 (1983), 242-253; solid phase directbiotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986),3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid phasedirect labeled assay, solid phase direct labeled sandwich assay; seeHarlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPress (1988); solid phase direct label RIA using I125 label; see Morelet al, Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand. J.Immunol. 32 (1990), 77-82. Typically, such an assay involves the use ofpurified tau or aggregates thereof bound to a solid surface or cellsbearing either of these, an unlabeled test immunoglobulin and a labeledreference immunoglobulin, i.e. the human monoclonal antibody of thepresent invention. Competitive inhibition is measured by determining theamount of label bound to the solid surface or cells in the presence ofthe test immunoglobulin. Usually the test immunoglobulin is present inexcess. In one embodiment, the competitive binding assay is performedunder conditions as described for the ELISA assay in the appendedExamples. Antibodies identified by competition assay (competingantibodies) include antibodies binding to the same epitope as thereference antibody and antibodies binding to an adjacent epitopesufficiently proximal to the epitope bound by the reference antibody forsteric hindrance to occur. Usually, when a competing antibody is presentin excess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50% or 75%. Hence, the present invention isfurther drawn to an antibody, or antigen-binding fragment, variant orderivatives thereof, where the antibody competitively inhibits areference antibody selected from the group consisting of NI-105.4E4,NI-105.24B2 or NI-105.4A3 from binding to tau.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH), where at least one ofVH-CDRs of the heavy chain variable region or at least two of theVH-CDRs of the heavy chain variable region are at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to reference heavy chain VH-CDR1,VH-CDR2 or VH-CDR3 amino acid sequences from the antibodies disclosedherein. Alternatively, the VH-CDR1, VH-CDR2 and VH-CDR3 regions of theVH are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toreference heavy chain VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequencesfrom the antibodies disclosed herein. Thus, according to this embodimenta heavy chain variable region of the invention has VH-CDR1, VH-CDR2 andVH-CDR3 polypeptide sequences related to the groups shown in FIG. 1.While FIG. 1 shows VH-CDRs defined by the Kabat system, other CDRdefinitions, e.g., VH-CDRs defined by the Chothia system, are alsoincluded in the present invention, and can be easily identified by aperson of ordinary skill in the art using the data presented in FIG. 1.In one embodiment, the amino acid sequence of the reference VH CDR1 isSEQ ID NO: 23, 29, or 35; the amino acid sequence of the reference VHCDR2 is SEQ ID NO: 24, 30 or 36; and the amino acid sequence of thereference VH CDR3 is SEQ ID NO: 25, 31 or 37.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH) in which the VH-CDR1,VH-CDR2 and VH-CDR3 regions have polypeptide sequences which areidentical to the VH-CDR1, VH-CDR2 and VH-CDR3 groups shown in FIG. 1. Inone embodiment, the amino acid sequence of the VH CDR1 is SEQ ID NO: 23,29, or 35; the amino acid sequence of the VH CDR2 is SEQ ID NO: 24, 30or 36; and the amino acid sequence of the VH CDR3 is SEQ ID NO: 25, 31or 37.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH) in which the VH-CDR1,VH-CDR2 and VH-CDR3 regions have polypeptide sequences which areidentical to the VH-CDR1, VH-CDR2 and VH-CDR3 groups shown in FIG. 1,except for one, two, three, four, five, six, seven, eight, nine, or tenamino acid substitutions in any one VH-CDR. In certain embodiments theamino acid substitutions are conservative. In one embodiment, the aminoacid sequence of the VH CDR1 is SEQ ID NO: 23, 29, or 35; the amino acidsequence of the VH CDR2 is SEQ ID NO: 24, 30 or 36; and the amino acidsequence of the VH CDR3 is SEQ ID NO: 25, 31 or 37.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (VL), where at least one ofthe VL-CDRs of the light chain variable region or at least two of theVL-CDRs of the light chain variable region are at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to reference light chain VL-CDR1,VL-CDR2 or VL-CDR3 amino acid sequences from antibodies disclosedherein. Alternatively, the VL-CDR1, VL-CDR2 and VL-CDR3 regions of theVL are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toreference light chain VL-CDR1, VL-CDR2 and VL-CDR3 amino acid sequencesfrom antibodies disclosed herein. Thus, according to this embodiment alight chain variable region of the invention has VL-CDR1, VL-CDR2 andVL-CDR3 polypeptide sequences related to the polypeptides shown inFIG. 1. While FIG. 1 shows VL-CDRs defined by the Kabat system, otherCDR definitions, e.g., VL-CDRs defined by the Chothia system, are alsoincluded in the present invention. In one embodiment, the amino acidsequence of the reference VL CDR1 is SEQ ID NO: 26, 32 or 38; the aminoacid sequence of the reference VL CDR2 is SEQ ID NO: 27, 33 or 39; andthe amino acid sequence of the reference VL CDR3 is SEQ ID NO: 28, 34 or40.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (VL) in which the VL-CDR1,VL-CDR2 and VL-CDR3 regions have polypeptide sequences which areidentical to the VL-CDR1, VL-CDR2 and VL-CDR3 groups shown in FIG. 1. Inone embodiment, the amino acid sequence of the VL CDR1 is SEQ ID NO: 26,32 or 38; the amino acid sequence of the VL CDR2 is SEQ ID NO: 27, 33 or39; and the amino acid sequence of the VL CDR3 is SEQ ID NO: 28, 34 or40.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VL) in which the VL-CDR1,VL-CDR2 and VL-CDR3 regions have polypeptide sequences which areidentical to the VL-CDR1, VL-CDR2 and VL-CDR3 groups shown in FIG. 1,except for one, two, three, four, five, six, seven, eight, nine, or tenamino acid substitutions in any one VL-CDR. In certain embodiments theamino acid substitutions are conservative. In one embodiment, the aminoacid sequence of the VL CDR1 is SEQ ID NO: 26, 32 or 38; the amino acidsequence of the VL CDR2 is SEQ ID NO: 27, 33 or 39; and the amino acidsequence of the VL CDR3 is SEQ ID NO: 28, 34 or 40.

An immunoglobulin or its encoding cDNA may be further modified. Thus, ina further embodiment the method of the present invention comprises anyone of the step(s) of producing a chimeric antibody, murinized antibody,single-chain antibody, Fab-fragment, bi-specific antibody, fusionantibody, labeled antibody or an analog of any one of those.Corresponding methods are known to the person skilled in the art and aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor (1988). When derivatives of saidantibodies are obtained by the phage display technique, surface plasmonresonance as employed in the BIAcore system can be used to increase theefficiency of phage antibodies which bind to the same epitope as that ofany one of the antibodies described herein (Schier, Human AntibodiesHybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995),7-13). The production of chimeric antibodies is described, for example,in international application WO89/09622. Methods for the production ofhumanized antibodies are described in, e.g., European application EP-A10 239 400 and international application WO90/07861. A further source ofantibodies to be utilized in accordance with the present invention areso-called xenogeneic antibodies. The general principle for theproduction of xenogeneic antibodies such as human-like antibodies inmice is described in, e.g., international applications WO91/10741,WO94/02602, WO96/34096 and WO 96/33735. As discussed above, the antibodyof the invention may exist in a variety of forms besides completeantibodies; including, for example, Fv, Fab and F(ab)2, as well as insingle chains; see e.g. international application WO88/09344.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as an immunostimulatoryligand at the carboxyl terminus; see, e.g., international applicationWO00/30680 for corresponding technical details.

Additionally, the present invention encompasses peptides including thosecontaining a binding molecule as described above, for example containingthe CDR3 region of the variable region of any one of the mentionedantibodies, in particular CDR3 of the heavy chain since it hasfrequently been observed that heavy chain CDR3 (HCDR3) is the regionhaving a greater degree of variability and a predominant participationin antigen-antibody interaction. Such peptides may easily be synthesizedor produced by recombinant means to produce a binding agent usefulaccording to the invention. Such methods are well known to those ofordinary skill in the art. Peptides can be synthesized for example,using automated peptide synthesizers which are commercially available.The peptides can also be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

Hence, the present invention relates to any binding molecule, e.g., anantibody or binding fragment thereof which is oriented towards the humananti-tau antibodies of the present invention and display the mentionedproperties, i.e. which specifically recognize tau. Such antibodies andbinding molecules can be tested for their binding specificity andaffinity by ELISA and Western Blot and immunohistochemistry as describedherein, see, e.g., the Examples. Furthermore, preliminary results ofsubsequent experiments performed in accordance with the presentinvention revealed that the human ant-tau antibody of the presentinvention, in particular antibody NI-105.4E4 binds primarily topathologically aggregated tau resembling neurofibrillary tangles (NFT),neuropil threads present on human brain sections of patients whosuffered from Alzheimer's disease (AD) in addition. Thus, in aparticular preferred embodiment of the present invention, the humanantibody or binding fragment, derivative or variant thereof recognizestau on human AD brain sections. Moreover, the distinct ability of theantibody NI-105.4E4 to differentially bind to tau pathologies could alsobe shown in transgenic mouse overexpressing human tau P301L. In additionto the already mentioned NFT's and neuropil threads the antibodyNI-105.4E4 binds on mouse brain sections also dystrophic neurites andidentifies tau aggregates at pre-tangle stage; see Example 4 and FIG. 6.

As an alternative to obtaining immunoglobulins directly from the cultureof immortalized B cells or B memory cells, the immortalized cells can beused as a source of rearranged heavy chain and light chain loci forsubsequent expression and/or genetic manipulation. Rearranged antibodygenes can be reverse transcribed from appropriate mRNAs to produce cDNA.If desired, the heavy chain constant region can be exchanged for that ofa different isotype or eliminated altogether. The variable regions canbe linked to encode single chain Fv regions. Multiple Fv regions can belinked to confer binding ability to more than one target or chimericheavy and light chain combinations can be employed. Once the geneticmaterial is available, design of analogs as described above which retainboth their ability to bind the desired target is straightforward.Methods for the cloning of antibody variable regions and generation ofrecombinant antibodies are known to the person skilled in the art andare described, for example, Gilliland et al., Tissue Antigens 47 (1996),1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells may be used; for efficient processing, however, mammaliancells may be considered. Typical mammalian cell lines useful for thispurpose include, but are not limited to, CHO cells, HEK 293 cells, orNSO cells.

The production of the antibody or analog is then undertaken by culturingthe modified recombinant host under culture conditions appropriate forthe growth of the host cells and the expression of the coding sequences.The antibodies are then recovered by isolating them from the culture.The expression systems are designed to include signal peptides so thatthe resulting antibodies are secreted into the medium; however,intracellular production is also possible.

In accordance with the above, the present invention also relates to apolynucleotide encoding the antibody or equivalent binding molecule ofthe present invention In one embodiment, the polynucleotide encodes atleast a variable region of an immunoglobulin chain of the antibodydescribed above. Typically, said variable region encoded by thepolynucleotide comprises at least one complementarity determining region(CDR) of the VH and/or VL of the variable region of the said antibody.

The person skilled in the art will readily appreciate that the variabledomain of the antibody having the above-described variable domain can beused for the construction of other polypeptides or antibodies of desiredspecificity and biological function. Thus, the present invention alsoencompasses polypeptides and antibodies comprising at least one CDR ofthe above-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art knowsthat binding affinity may be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs asdefined by Kabat; see, e.g., Riechmann, et al, Nature 332 (1988),323-327. Thus, the present invention also relates to antibodies whereinone or more of the mentioned CDRs comprise one or more, or not more thantwo amino acid substitutions. In one embodiment, the antibody of theinvention comprises in one or both of its immunoglobulin chains two orall three CDRs of the variable regions as set forth in FIG. 1.

Binding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention, as known by those ofordinary skill in the art, can comprise a constant region which mediatesone or more effector functions. For example, binding of the C1 componentof complement to an antibody constant region may activate the complementsystem. Activation of complement is important in the opsonization andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region, with a Fc receptor binding site on the antibody Fcregion binding to a Fc receptor (FcR) on a cell. There are a number ofFc receptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

Accordingly, certain embodiments of the present invention include anantibody, or antigen-binding fragment, variant, or derivative thereof,in which at least a fraction of one or more of the constant regiondomains has been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of tau aggregation and deposition, reduced serum half-life, orincreased serum half-life when compared with a whole, unaltered antibodyof approximately the same immunogenicity. For example, certainantibodies for use in the diagnostic and treatment methods describedherein are domain deleted antibodies which comprise a polypeptide chainsimilar to an immunoglobulin heavy chain, but which lack at least aportion of one or more heavy chain domains. For instance, in certainantibodies, one entire domain of the constant region of the modifiedantibody will be deleted, for example, all or part of the CH2 domainwill be deleted. In other embodiments, certain antibodies for use in thediagnostic and treatment methods described herein have a constantregion, e.g., an IgG heavy chain constant region, which is altered toeliminate glycosylation, referred to elsewhere herein as aglycosylatedor “agly” antibodies. Such “agly” antibodies may be preparedenzymatically as well as by engineering the consensus glycosylationsite(s) in the constant region. While not being bound by theory, it isbelieved that “agly” antibodies may have an improved safety andstability profile in vivo. Methods of producing aglycosylatedantibodies, having desired effector function are found for example ininternational application WO2005/018572, which is incorporated byreference in its entirety.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tau localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as taulocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well know immunological techniques withoutundue experimentation.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences to increase the cellularuptake of antibodies by way of example by enhancing receptor-mediatedendocytosis of antibodies via Fcγ receptors, LRP, or Thy1 receptors orby ‘SuperAntibody Technology’, which is said to enable antibodies to beshuttled into living cells without harming them (Expert Opin. Biol.Ther. (2005), 237-241). For example, the generation of fusion proteinsof the antibody binding region and the cognate protein ligands of cellsurface receptors or bi- or multi-specific antibodies with a specificsequences biding to tau as well as a cell surface receptor may beengineered using techniques known in the art.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences or the antibody may bechemically modified to increase its blood brain barrier penetration.

Modified forms of antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be made from whole precursor orparent antibodies using techniques known in the art. Exemplarytechniques are discussed in more detail herein. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be made or manufactured using techniques that are known inthe art. In certain embodiments, antibody molecules or fragments thereofare “recombinantly produced,” i.e., are produced using recombinant DNAtechnology. Exemplary techniques for making antibody molecules orfragments thereof are discussed in more detail elsewhere herein.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention also include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In particular embodiments, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention will not elicit adeleterious immune response in the animal to be treated, e.g., in ahuman. In certain embodiments, binding molecules, e.g., antibodies, orantigen-binding fragments thereof of the invention are derived from apatient, e.g., a human patient, and are subsequently used in the samespecies from which they are derived, e.g., human, alleviating orminimizing the occurrence of deleterious immune responses.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes; see, e.g., internationalapplications WO98/52976 and WO00/34317. For example, VH and VL sequencesfrom the starting antibody are analyzed and a human T cell epitope “map”from each V region showing the location of epitopes in relation tocomplementarity determining regions (CDRs) and other key residues withinthe sequence. Individual T cell epitopes from the T cell epitope map areanalyzed in order to identify alternative amino acid substitutions witha low risk of altering activity of the final antibody. A range ofalternative VH and VL sequences are designed comprising combinations ofamino acid substitutions and these sequences are subsequentlyincorporated into a range of binding polypeptides, e.g., tau-specificantibodies or immunospecific fragments thereof for use in the diagnosticand treatment methods disclosed herein, which are then tested forfunction. Typically, between 12 and 24 variant antibodies are generatedand tested. Complete heavy and light chain genes comprising modified Vand human C regions are then cloned into expression vectors and thesubsequent plasmids introduced into cell lines for the production ofwhole antibody. The antibodies are then compared in appropriatebiochemical and biological assays, and the optimal variant isidentified.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981), said references incorporatedby reference in their entireties. The term “monoclonal antibody” as usedherein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. In certain embodiments, antibodies of the presentinvention are derived from human B cells which have been immortalizedvia transformation with Epstein-Barr virus, as described herein.

In the well-known hybridoma process (Kohler et al., Nature 256 (1975),495) the relatively short-lived, or mortal, lymphocytes from a mammal,e.g., B cells derived from a human subject as described herein, arefused with an immortal tumor cell line (e.g., a myeloma cell line),thus, producing hybrid cells or “hybridomas” which are both immortal andcapable of producing the genetically coded antibody of the B cell. Theresulting hybrids are segregated into single genetic strains byselection, dilution, and re-growth with each individual straincomprising specific genes for the formation of a single antibody. Theyproduce antibodies, which are homogeneous against a desired antigen and,in reference to their pure genetic parentage, are termed “monoclonal”.

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that contain one or more substances that inhibit the growth orsurvival of the unfused, parental myeloma cells. Those skilled in theart will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. The binding specificity of the monoclonal antibodiesproduced by hybridoma cells is determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA) as described herein. After hybridoma cellsare identified that produce antibodies of the desired specificity,affinity and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods; see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, pp59-103 (1986). It will further be appreciated that the monoclonalantibodies secreted by the subclones may be separated from culturemedium, ascites fluid or serum by conventional purification proceduressuch as, for example, protein-A, hydroxylapatite chromatography, gelelectrophoresis, dialysis or affinity chromatography.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized or naturally immunemammal, e.g., a human, and cultured for about 7 days in vitro. Thecultures can be screened for specific IgGs that meet the screeningcriteria. Cells from positive wells can be isolated. IndividualIg-producing B cells can be isolated by FACS or by identifying them in acomplement-mediated hemolytic plaque assay. Ig-producing B cells can bemicromanipulated into a tube and the VH and VL genes can be amplifiedusing, e.g., RT-PCR. The VH and VL genes can be cloned into an antibodyexpression vector and transfected into cells (e.g., eukaryotic orprokaryotic cells) for expression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments may be producedrecombinantly or by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)2 fragments). F(ab′)2 fragments contain the variableregion, the light chain constant region and the CH1 domain of the heavychain. Such fragments are sufficient for use, for example, inimmunodiagnostic procedures involving coupling the immunospecificportions of immunoglobulins to detecting reagents such as radioisotopes.

Human antibodies, such as described herein, are particularly desirablefor therapeutic use in human patients. Human antibodies of the presentinvention are isolated, e.g., from healthy human subjects who because oftheir age may be suspected to be at risk of developing a tauopathicdisorder, e.g., Alzheimer's disease, or a patient with the disorder butwith an unusually stable disease course. However, though it is prudentto expect that elderly healthy and symptom-free subjects, respectively,more regularly will have developed protective anti-tau antibodies thanyounger subjects, the latter may be used as well as source for obtaininga human antibody of the present invention. This is particularly true foryounger patients who are predisposed to develop a familial form of atauopathic disease but remain symptom-free since their immune systemfunctions more efficiently than that in older adults.

In one embodiment, an antibody of the invention comprises at least oneheavy or light chain CDR of an antibody molecule. In another embodiment,an antibody of the invention comprises at least two CDRs from one ormore antibody molecules. In another embodiment, an antibody of theinvention comprises at least three CDRs from one or more antibodymolecules. In another embodiment, an antibody of the invention comprisesat least four CDRs from one or more antibody molecules. In anotherembodiment, an antibody of the invention comprises at least five CDRsfrom one or more antibody molecules. In another embodiment, an antibodyof the invention comprises at least six CDRs from one or more antibodymolecules. Exemplary antibody molecules comprising at least one CDR thatcan be included in the subject antibodies are described herein.

Antibodies of the present invention can be produced by any method knownin the art for the synthesis of antibodies, in particular, by chemicalsynthesis or by recombinant expression techniques as described herein.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises a synthetic constantregion wherein one or more domains are partially or entirely deleted(“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire CH2 domain has been removed (ΔCH2 constructs). Forother embodiments a short connecting peptide may be substituted for thedeleted domain to provide flexibility and freedom of movement for thevariable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the CH2 domain on the catabolic rate of the antibody. Domain deletedconstructs can be derived using a vector encoding an IgG1 human constantdomain, see, e.g., international applications WO02/060955 andWO02/096948A2. This vector is engineered to delete the CH2 domain andprovide a synthetic vector expressing a domain deleted IgG1 constantregion.

In certain embodiments, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the present invention areminibodies. Minibodies can be made using methods described in the art,see, e.g., U.S. Pat. No. 5,837,821 or international application WO94/09817.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises an immunoglobulin heavychain having deletion or substitution of a few or even a single aminoacid as long as it permits association between the monomeric subunits.For example, the mutation of a single amino acid in selected areas ofthe CH2 domain may be enough to substantially reduce Fc binding andthereby increase tau localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement binding) to be modulated.Such partial deletions of the constant regions may improve selectedcharacteristics of the antibody (serum half-life) while leaving otherdesirable functions associated with the subject constant region domainintact. Moreover, as alluded to above, the constant regions of thedisclosed antibodies may be synthetic through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it may be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

The present invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the VH regions and/or VL regions) describedherein, which antibodies or fragments thereof immunospecifically bind totau. Standard techniques known to those of skill in the art can be usedto introduce mutations in the nucleotide sequence encoding an antibody,including, but not limited to, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions. Inone embodiment, the variants (including derivatives) encode less than 50amino acid substitutions, less than 40 amino acid substitutions, lessthan 30 amino acid substitutions, less than 25 amino acid substitutions,less than 20 amino acid substitutions, less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the reference VH region, VH-CDR1, VH-CDR2, VH-CDR3, VLregion, VL-CDR1, VL-CDR2, or VL-CDR3. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a side chain with a similar charge. Familiesof amino acid residues having side chains with similar charges have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity (e.g., the ability to bind tau).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, e.g., have no, orlittle, effect on an antibody's ability to bind antigen, indeed somesuch mutations do not alter the amino acid sequence whatsoever. Thesetypes of mutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Codon-optimized coding regions encodingantibodies of the present invention are disclosed elsewhere herein.Alternatively, non-neutral missense mutations may alter an antibody'sability to bind antigen. The location of most silent and neutralmissense mutations is likely to be in the framework regions, while thelocation of most non-neutral missense mutations is likely to be in CDR,though this is not an absolute requirement. One of skill in the artwould be able to design and test mutant molecules with desiredproperties such as no alteration in antigen-binding activity oralteration in binding activity (e.g., improvements in antigen-bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein may routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of tau) can be determinedusing techniques described herein or by routinely modifying techniquesknown in the art.

Tau binding agents, for example, but not limited to, tau bindingantibodies of the present invention may be characterized using any invivo or in vitro models of neurodegenerative tauopathies. A skilledartisan readily understands that a tau binding agent (e.g., an antibody)of the invention may be characterized in a mouse model forneurodegenerative tauopathies, for example, but not limited to, any oneof the following three different animal models for tauopathies may beused to characterize and validate the tau antibodies (and molecules withthe binding specificities thereof) of the present invention.

1. Transgenic TauP301L mice (line183): expressing human Tau40 with P301Lmutation under the murine Thy1.2 promoter (Generation of thesetransgenic animals is described in Götz et al., J. Biol. Chem. 276(2001), 529-534 and in international application WO 2003/017918, thedisclosure content of which is incorporated herein by reference)

2. JNPL3 mice expressing the shortest human tau isoform with P301Lmutation under the murine PrP promoter (available from Taconic, Hudson,N.Y., U.S.A).

3. P301STau (line PS19) mice expressing human tau with P301S mutationunder the murine PrP promoter (available from the Jackson Laboratory,Bar Harbor, Me., U.S.A).

A skilled artisan understands that an experimental model ofneurodegenerative tauopathies may be used in a preventative setting orit may be used in a therapeutic setting. In a preventative setting, thedosing of animals starts prior to the onset of the neurodegenerativetauopathies or symptoms thereof. In preventative settings, a tau bindingagent (e.g., antibody) of the invention is evaluated for its ability toprevent, reduce or delay the onset of neurodegenerative tauopathies orsymptoms thereof. In a therapeutic setting, the dosing of animals startafter the onset of neurodegenerative tauopathies or a symptom thereof.In a therapeutic setting, a tau binding agent (e.g., antibody) of theinvention is evaluated for its ability to treat, reduce or alleviate theneurodegenerative tauopathies or a symptom thereof. Symptoms of theneurodegenerative tauopathies include, but are not limited to,accumulation of pathological tau deposits, neurofibrillary tangles(NFT), hyperphosphorylated tau polypeptide, insoluble tau fractions inthe neurons, brain, spinal cord, cerebrospinal fluid or serum of theexperimental object. A skilled artisan understands that a positivepreventative or therapeutic outcome in any animal model ofneurodegenerative tauopathies indicates that the particular tau bindingagent (e.g., antibody) may be used for preventative or therapeuticpurposes in a subject other than the experimental model organism, forexample, it may be used to treat neurodegenerative tauopathies in ahuman subject in need thereof.

In one embodiment, a tau binding agent (e.g., an antibody) of theinvention may be administered to a tauopathy mouse model andcorresponding control wild type mice. The antibody administered may be amurinized antibody of the present invention or a human-murine chimera ofan antibody of the present invention. See, for example, Example 6 and 7.The tau binding agent (e.g., an antibody) may be administered by anymeans known in the art, for example, by intraperitoneal, intracranial,intramuscular, intravenous, subcutaneous, oral, and aerosoladministration. Experimental animals may be given one, two, three, four,five or more doses of the tau binding agent (e.g., an antibody) or acontrol composition, such as PBS. In one embodiment, experimentalanimals will be administered one or two doses of a tau binding agent(e.g., an antibody). See, for example, Example 9. In another embodiment,the animals are chronically dosed with the tau binding agent (e.g., anantibody) over several weeks or months. See, for example, Example 10. Askilled artisan can readily design a dosing regimen that fits theexperimental purpose, for example, dosing regimen for acute studies,dosing regimen for chronic studies, dosing regimen for toxicity studies,dosing regimen for preventative or therapeutic studies. The presence ofthe tau binding agent (e.g., antibody) in a particular tissuecompartment of the experimental animals, for example, but not limitedto, serum, blood, cerebrospinal fluid, brain tissue, may be establishedusing well know methods of the art. See, for example, Example 9 and 10.In one embodiment, a tau binding agent (e.g., antibody) of the inventionis capable to penetrate the blood brain barrier. A skilled artisanunderstands that by adjusting the tau binding agent (e.g., antibody)dose and the dosing frequency, a desired tau binding agent (e.g.,antibody) concentration may be maintained in the experimental animals.Any effect of a tau binding agent (e.g., antibody) of the presentinvention in the tauopathy models may be assessed by comparing thelevel, biochemical characteristics or distribution of tau in the treatedand control animals. In one example, the neurofibrillary tangles (NFT)are examined using the silver impregnation technique of Gallyas or byimmunostaining with monoclonal mouse antibody AT100 and AT180, whichrecognize pathologically phosphorylated tau in NFT. The number orfrequency of Gallyas-positive neurons and/or AT100, AT180 labeledneurons in the brain and spinal cord in antibody treated mice andcontrol animals may be determined to evaluate the effect of antibodytreatment. In one embodiment, an antibody of the present invention iscapable of reducing the level, amount or concentration ofneurofibrillary tangles in the brain or spinal cord in an animal model.The antibody may reduce the level, amount or concentration ofneurofibrillary tangles by at least about 5%, 10%, 20%, 30%, 50%, 70%,90% or more. In another embodiment, an antibody of the present inventionis capable of reducing the number or frequency of Gallyas-positiveneurons in the brain or spinal cord in an animal model, for example, byat least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. In a furtherembodiment, an antibody of the present invention is capable of reducingthe number or frequency of AT100 or AT180 antibody positive neurons inthe brain or spinal cord in an animal model, for example, by at leastabout 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. The effect of anantibody of the present invention may also be assessed by examining thedistribution and biochemical properties of tau following antibodyadministration. In one embodiment, an antibody of the present inventionis capable of reducing the amount or concentration of tau protein in thebrain or spinal cord of an animal model, for example, by at least about5%, 10%, 20%, 30%, 50%, 70%, 90% or more. In another embodiment, anantibody of the present invention is capable of reducing the amount orconcentration of insoluble tau protein in the brain or spinal cord of ananimal model, for example, by at least about 5%, 10%, 20%, 30%, 50%,70%, 90% or more. Insoluble tau fraction may be prepared as described,for example, in Example 10 or in Goedert M, Spillantini M G, Cairns N J,Crowther R A. Neuron 8, 159 (1992). The amount of tau protein in abiological sample may be determined by any method known to one of skill,for example, as described in Example 10. In a further embodiment, anantibody of the present invention may reduce the amount or concentrationof hyperphosphorylated tau protein in the brain or spinal cord in ananimal model, for example, by at least about 5%, 10%, 20%, 30%, 50%,70%, 90% or more. Hyperphosphorylated tau may be detected usingantibodies specific for pathologically hyperphosphorylated forms of tau,such as AT100 or AT180. An antibody of the present invention may alsoreduce tau concentration in the blood, serum or cerebrospinal fluid oran animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%,70%, 90% or more. In one embodiment, the % reduction is relativecompared to the level, number, frequency, amount or concentration thatexisted before treatment, or to the level, number, frequency, amount orconcentration that exist in an untreated/control treated subject.

In one embodiment, an antibody of the present invention may prevent ordelay the onset of at least one symptom of a neurodegenerative tauopathyin a subject. In one embodiment, an antibody of the present inventionmay reduce or eliminate at least one symptom of a neurodegenerativetauopathy in a subject. The symptom may be the formation of pathologicaltau deposits, hyperphosphorylated tau deposits, insoluble tau deposits,neurofibrillary fibers, neurofibrillary fibers, pre-tangle phosphor tauaggregates, intraneuronal neurofibrillary tangles or extraneuronalneurofibrillary tangles in the brain or spinal cord of a subject. See,e.g., Augustinack et al, Acta Neuropathol 103:26-35 (2002). The symptommay also be the presence, or elevated concentration or amount, of tau inthe serum, blood, urine or cerebrospinal fluid, wherein elevatedconcentration amount is compared to a healthy subject. The symptom maybe a neurological symptom, for example, altered conditioned tasteaversion, altered contextual fear conditioning, memory impairment, lossof motor function. In one embodiment, memory impairment is assessedusing a two-trial Y-maze task. In one embodiment, the at least onesymptom is reduced by at least about 5%, 10%, 15%, 20%, 30%, 50%, 70%,or 90%. The present invention also provides a method of preventing ordelaying the onset of at least one symptom of a neurodegenerativetauopathy in a subject in need thereof, comprising administering atherapeutically effective amount of a tau antibody described herein. Thepresent invention further provides a method of reducing or eliminatingleast one symptom of a neurodegenerative tauopathy in a subject in needthereof, comprising administering a therapeutically effective amount ofa tau antibody described herein. In one embodiment, the subject is anexperimental organism, such as, but not limited to, transgenic mouse. Inone embodiment, the subject is a human.

III. Polynucleotides Encoding Antibodies

A polynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of any polyribonucleotideor polydeoxribonucleotide, which may be unmodified RNA or DNA ormodified RNA or DNA. For example, a polynucleotide encoding an antibody,or antigen-binding fragment, variant, or derivative thereof can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, a polynucleotide encoding an antibody, orantigen-binding fragment, variant, or derivative thereof can be composedof triple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis. In oneembodiment, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

As is well known, RNA may be isolated from the original B cells,hybridoma cells or from other transformed cells by standard techniques,such as guanidinium isothiocyanate extraction and precipitation followedby centrifugation or chromatography. Where desirable, mRNA may beisolated from total RNA by standard techniques such as chromatography onoligo dT cellulose. Suitable techniques are familiar in the art. In oneembodiment, cDNAs that encode the light and the heavy chains of theantibody may be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as human constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(VH), where at least one of the CDRs of the heavy chain variable regionor at least two of the VH-CDRs of the heavy chain variable region are atleast 80%, 85%, 90%, 95%, 96%, about 97%, 98%, or 99% identical toreference heavy chain VH-CDR1, VH-CDR2, or VH-CDR3 amino acid sequencesfrom the antibodies disclosed herein. Alternatively, the VH-CDR1,VH-CDR2, or VH-CDR3 regions of the VH are at least 80%, 85%, 90%, 95%,96%, about 97%, 98%, or 99% identical to reference heavy chain VH-CDR1,VH-CDR2, and VH-CDR3 amino acid sequences from the antibodies disclosedherein. Thus, according to this embodiment a heavy chain variable regionof the invention has VH-CDR1, VH-CDR2, or VH-CDR3 polypeptide sequencesrelated to the polypeptide sequences shown in FIG. 1. In one embodiment,the amino acid sequence of the reference VH CDR1 is SEQ ID NO: 23, 29,or 35; the amino acid sequence of the reference VH CDR2 is SEQ ID NO:24, 30 or 36; and the amino acid sequence of the reference VH CDR3 isSEQ ID NO: 25, 31 or 37.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(VH), in which the VH-CDR1, VH-CDR2 and VH-CDR3 regions have polypeptidesequences which are identical to the VH-CDR1, VH-CDR2 and VH-CDR3 groupsshown in FIG. 1, except for one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions in any one VH-CDR. Incertain embodiments the amino acid substitutions are conservative. Inone embodiment, the amino acid sequence of the VH CDR1 is SEQ ID NO: 23,29, or 35; the amino acid sequence of the VH CDR2 is SEQ ID NO: 24, 30or 36; and the amino acid sequence of the VH CDR3 is SEQ ID NO: 25, 31or 37.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region(VL), where at least one of the VL-CDRs of the light chain variableregion or at least two of the VL-CDRs of the light chain variable regionare at least 80%, 85%, 90%, 95%, 96%, about 97%, 98%, or 99% identicalto reference light chain VL-CDR1, VL-CDR2, or VL-CDR3 amino acidsequences from the antibodies disclosed herein. Alternatively, theVL-CDR1, VL-CDR2, or VL-CDR3 regions of the VL are at least 80%, 85%,90%, 95%, 96%, about 97%, 98%, or 99% identical to reference light chainVL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences from the antibodiesdisclosed herein. Thus, according to this embodiment a light chainvariable region of the invention has VL-CDR1, VL-CDR2, or VL-CDR3polypeptide sequences related to the polypeptide sequences shown inFIG. 1. In one embodiment, the amino acid sequence of the reference VLCDR1 is SEQ ID NO: 26, 32 or 38; the amino acid sequence of thereference VL CDR2 is SEQ ID NO: 27, 33 or 39; and the amino acidsequence of the reference VL CDR3 is SEQ ID NO: 28, 34 or 40.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region (VL)in which the VL-CDR1, VL-CDR2 and VL-CDR3 regions have polypeptidesequences which are identical to the VL-CDR1, VL-CDR2 and VL-CDR3 groupsshown in FIG. 1, except for one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions in any one VL-CDR. Incertain embodiments the amino acid substitutions are conservative. Inone embodiment, the amino acid sequence of the VL CDR1 is SEQ ID NO: 26,32 or 38; the amino acid sequence of the VL CDR2 is SEQ ID NO: 27, 33 or39; and the amino acid sequence of the VL CDR3 is SEQ ID NO: 28, 34 or40.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region (VH)in which the VH-CDR1, VH-CDR2, and VH-CDR3 regions have polypeptidesequences which are identical to the VH-CDR1, VH-CDR2, and VH-CDR3groups shown in FIG. 1. In one embodiment, the amino acid sequence ofthe VH CDR1 is SEQ ID NO: 23, 29, or 35; the amino acid sequence of theVH CDR2 is SEQ ID NO: 24, 30 or 36; and the amino acid sequence of theVH CDR3 is SEQ ID NO: 25, 31 or 37.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region (VL)in which the VL-CDR1, VL-CDR2, and VL-CDR3 regions have polypeptidesequences which are identical to the VL-CDR1, VL-CDR2, and VL-CDR3groups shown in FIG. 1. In one embodiment, the amino acid sequence ofthe VL CDR1 is SEQ ID NO: 26, 32 or 38; the amino acid sequence of theVL CDR2 is SEQ ID NO: 27, 33 or 39; and the amino acid sequence of theVL CDR3 is SEQ ID NO: 28, 34 or 40.

As known in the art, “sequence identity” between two polypeptides or twopolynucleotides is determined by comparing the amino acid or nucleicacid sequence of one polypeptide or polynucleotide to the sequence of asecond polypeptide or polynucleotide. When discussed herein, whether anyparticular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2 (1981), 482-489, to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

In one embodiment of the present invention, the polynucleotidecomprises, consists essentially of, or consists of a nucleic acid havinga polynucleotide sequence of the VH or VL region of an anti-tau antibodyas depicted in Table 2. In this respect, the person skilled in the artwill readily appreciate that the polynucleotides encoding at least thevariable domain of the light and/or heavy chain may encode the variabledomain of both immunoglobulin chains or only one. The present inventionfurther provides a polynucleotide comprising, or consisting of anucleotide sequence encoding the amino acid sequence of SEQ ID: 93.

TABLE 2 Nucleotide sequences of the V_(H) and V_(L) region of tauspecific antibodies. Nucleotide sequences of variable heavy (VH) Antibody and variable light (VL) chains NI-105.4E4-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGATC SEQ ID NO: 8CCTGAAACTCTCCTGTGCAGCCTCTGGGTTCAATTTCAACATCTCTGCTATACACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTTGGCCGAATAAGAAGTAAATCTCACAATTACGCGACTTTATATGCTGCGTCCCTGAAAGGCCGGTTCACCCTCTCCAGAGATGATTCAAGGAACACGGCGTATCTGCAAATGAGCAGCCTGCAAACCGAGGATATGGCCGICTATTACTGTACTGITCTGAGTGCGAATTACGACACCTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCCTCGNI-105.4E4-V_(L) TCCTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACSEQ ID NO: 10 GGCCAGGATCTCCTGCTTTGGAGATACATTGCCAAAGCAATATACTTATTGGTATCAGCAGAAGCCTGGCCAGGCCCCTGTGTTAGTGATTTATAAAGACACTGAGAGGCCCTCAGGGATCCCCGAGCGATTCTCTGGCTCCAGCTCAGGGACAACAGTCACCTTGACCATCAGTGGAGTCCAGGCAGAAGACGAGGCTGACTATTACTGTCTATCAGCTGACAACAGTGCTACTTGGGTGTTCGGCGGA GGGACCAAGGTGACCGTCCTANI-105.24B2-V_(H) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCSEQ ID NO: 12 GGTGAAGGTTTCCTGTAAGGCATCTGGATACACCTTCGTCAATTACATTATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATCATCAATCCTAATGGCGGAAACACAAGTTATGCAGAGAAATTCCAGGCCCGAGTCACCTTGACCAGCGACACGTCTACGAGTACGGTGTACATGGACCTGAGCAGCCTGACATCTGAGGACACGGCCGTCTATTACTGTGCCGTCCTTTCCCCTTCGAATCCCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCG NI-105.24B2-V_(L)TCCTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGAC SEQ ID NO: 14GGCCGGGATCACCTGCTCTGGAGATGCTTTGCCAAAGCAATTTGTTTATTGGTACCAGAAGAAGCCAGGCCAGGCCCCTGTGTTATTGATATATAAAGACACTGAGAGGCCCTCACGAATCCCTGAGCGCTTCTCTGGCTCCACCTCAGGGACAACAGTCGCGTTGACCATCAATGGGGTCCAGGCAGAGGACGAGGCTGACTATTACTGTCAATCAGCCGACCGCAGTGGTGCTCTTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA NI-105.4A3-V_(H)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGCGGTCCAGCCTGGGGGGTC SEQ ID NO: 16CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTATGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGCAGTGGGTGGCAGTTATATCGTATGAGGGAACTTATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGAACTTGCAGATGAGCAGCCTGAGAGTTGAAGACACGGCTGTGTATTTCTGTGTGAAAGCTCGAGCCTTTGCCTCCGGACAGCGAAGCACCTCCACCGTACCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG NI-105.4A3-V_(L)TCCTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAAAC SEQ ID NO: 18GGCCAGGATCACCTGCTCTGGAGATGCATTGCCAAAAAAATATGCTTATTGGTACCAGCAGAAGTCAGGCCAGGCCCCTGTGTTGGTCATCTATGAGGACAACAAACGACCCTCCGGGATCCCTGAGAGATTCTCTGGCTCCAGCTCAGGGACAGTGGCCACCTTGACTATCAGTGGGGCCCAGGTGGACGATGAAGCTGACTACTACTGCTACTCGACAGACATCAGTGGTGACCTTCGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTC

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region atleast 80%, 85%, 90%, 95%, 96%, about 97%, 98%, or 99% or 95% identicalto reference heavy chain VH. In one embodiment, the amino acid sequenceof the reference heavy chain variable region is SEQ ID NO: 9, 13, 17 or93.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region atleast 80%, 85%, 90%, 95%, 96%, about 97%, 98%, or 99% or 95% identicalto reference light chain VL. In one embodiment, the amino acid sequenceof the reference light chain variable region is SEQ ID NO: 11, 15 or 19.

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotideswhich encode fusion polynucleotides, Fab fragments, and otherderivatives, as described herein, are also contemplated by theinvention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides, e.g., as described in Kutmeieret al., BioTechniques 17 (1994), 242, which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an antibody, or antigen-bindingfragment, variant, or derivative thereof may be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may bechemically synthesized or obtained from a suitable source (e.g., anantibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA+ RNA, isolated from, any tissue or cellsexpressing the tau-specific antibody, such as hybridoma cells selectedto express an antibody) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody, or antigen-binding fragment, variant, or derivativethereof is determined, its nucleotide sequence may be manipulated usingmethods well known in the art for the manipulation of nucleotidesequences, e.g., recombinant DNA techniques, site directed mutagenesis,PCR, etc. (see, for example, the techniques described in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

IV. Expression of Antibody Polypeptides

Following manipulation of the isolated genetic material to provideantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, the polynucleotides encoding the antibodiesare typically inserted in an expression vector for introduction intohost cells that may be used to produce the desired quantity of antibody.Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody which binds to atarget molecule is described herein. Once a polynucleotide encoding anantibody molecule or a heavy or light chain of an antibody, or portionthereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,international applications WO 86/05807 and WO 89/01036; and U.S. Pat.No. 5,122,464) and the variable domain of the antibody may be clonedinto such a vector for expression of the entire heavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells. For the purposes of this invention, numerousexpression vector systems may be employed. For example, one class ofvector utilizes DNA elements which are derived from animal viruses suchas bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus,baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Othersinvolve the use of polycistronic systems with internal ribosome bindingsites. Additionally, cells which have integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow selection of transfected host cells. The marker may provide forprototrophy to an auxotrophic host, biocide resistance (e.g.,antibiotics) or resistance to heavy metals such as copper. Theselectable marker gene can either be directly linked to the DNAsequences to be expressed, or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include signal sequences, splicesignals, as well as transcriptional promoters, enhancers, andtermination signals.

In particular embodiments the cloned variable region genes are insertedinto an expression vector along with the heavy and light chain constantregion genes (e.g., human heavy and light chain constant region genes)as discussed above. In one embodiment, this is effected using aproprietary expression vector of Biogen IDEC, Inc., referred to asNEOSPLA, disclosed in U.S. Pat. No. 6,159,730. This vector contains thecytomegalovirus promoter/enhancer, the mouse beta globin major promoter,the SV40 origin of replication, the bovine growth hormonepolyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2,the dihydrofolate reductase gene and leader sequence. This vector hasbeen found to result in very high level expression of antibodies uponincorporation of variable and constant region genes, transfection in CHOcells, followed by selection in G418 containing medium and methotrexateamplification. Of course, any expression vector which is capable ofeliciting expression in eukaryotic cells may be used in the presentinvention. Examples of suitable vectors include, but are not limited toplasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2,pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (availablefrom Invitrogen, San Diego, Calif.), and plasmid pCI (available fromPromega, Madison, Wis.). In general, screening large numbers oftransformed cells for those which express suitably high levels ifimmunoglobulin heavy and light chains is routine experimentation whichcan be carried out, for example, by robotic systems. Vector systems arealso taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which isincorporated by reference in its entirety herein. This system providesfor high expression levels, e.g., >30 pg/cell/day. Other exemplaryvector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.

In other embodiments the antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be expressed usingpolycistronic constructs such as those disclosed in US patentapplication publication no. 2003-0157641 A1 and incorporated herein inits entirety. In these expression systems, multiple gene products ofinterest such as heavy and light chains of antibodies may be producedfrom a single polycistronic construct. These systems advantageously usean internal ribosome entry site (IRES) to provide relatively high levelsof antibodies. Compatible IRES sequences are disclosed in U.S. Pat. No.6,193,980 which is also incorporated herein. Those skilled in the artwill appreciate that such expression systems may be used to effectivelyproduce the full range of antibodies disclosed in the instantapplication.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the antibody has been prepared, the expression vector may beintroduced into an appropriate host cell. Introduction of the plasmidinto the host cell can be accomplished by various techniques well knownto those of skill in the art. These include, but are not limited to,transfection including lipotransfection using, e.g., Fugene® orlipofectamine, protoplast fusion, calcium phosphate precipitation, cellfusion with enveloped DNA, microinjection, and infection with intactvirus. Typically, plasmid introduction into the host is via standardcalcium phosphate co-precipitation method. The host cells harboring theexpression construct are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inparticular embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad.Sci. USA 77 (1980), 2197. The coding sequences for the heavy and lightchains may comprise cDNA or genomic DNA.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, NSO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). In one embodiment, bacterial cellssuch as Escherichia coli, and more preferably, eukaryotic cells,especially for the expression of whole recombinant antibody molecule,are used for the expression of a recombinant antibody molecule. Forexample, mammalian cells such as Chinese Hamster Ovary (CHO) cells, inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies; see, e.g., Foecking et al., Gene 45 (1986), 101;Cockett et al., Bio/Technology 8 (1990), 2.

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability to determineparticular host cell lines which are best suited for the desired geneproduct to be expressed therein. Exemplary host cell lines include, butare not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB11(Chinese Hamster Ovary lines, DHFR minus), HELA (human cervicalcarcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinesehamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidneyline), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT(bovine endothelial cells), RAJI (human lymphocyte) and 293 (humankidney). In a specific embodiment, host cell lines are CHO or 293 cells.Host cell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska& Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adeninephosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl.Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78(1981), 2072); neo, which confers resistance to the aminoglycoside G-418Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu,Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32(1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan andAnderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993),155-215; and hygro, which confers resistance to hygromycin (Santerre etal., Gene 30 (1984), 147. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons,NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al.(eds), Current Protocols in Human Genetics, John Wiley & Sons, NY(1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification, for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase; see Crouse et al., Mol. Cell. Biol. 3(1983), 257.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can also be expressed innon-mammalian cells such as bacteria or insect or yeast or plant cells.Bacteria which readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies; see, e.g., internationalapplication WO02/096948.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983),1791), in which the antibody coding sequence may be ligated individuallyinto the vector in frame with the lacZ coding region so that a fusionprotein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13(1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989),5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to a matrix ofglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris. For expression inSaccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al.,Gene 10 (1980), 157) is commonly used. This plasmid already contains theTRP1 gene which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example ATCC No. 44076 orPEP4-1 (Jones, Genetics 85 (1977), 12). The presence of the trpl lesionas a characteristic of the yeast host cell genome then provides aneffective environment for detecting transformation by growth in theabsence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingfor example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,e.g. ammonium sulfate precipitation, or by any other standard techniquefor the purification of proteins; see, e.g., Scopes, “ProteinPurification”, Springer Verlag, N.Y. (1982). Alternatively, anothermethod for increasing the affinity of antibodies of the invention isdisclosed in US patent publication 2002-0123057 A1.

V. Fusion Proteins and Conjugates

In certain embodiments, the antibody polypeptide comprises an amino acidsequence or one or more moieties not normally associated with anantibody. Exemplary modifications are described in more detail below.For example, a single-chain fv antibody fragment of the invention maycomprise a flexible linker sequence, or may be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label such as afluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and thelike)

An antibody polypeptide of the invention may comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulintau-binding domain with at least one target binding site, and at leastone heterologous portion, i.e., a portion with which it is not naturallylinked in nature. The amino acid sequences may normally exist inseparate proteins that are brought together in the fusion polypeptide orthey may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to an antibody, or an antigen-binding fragment, variant, oranalog thereof is derived from a non-immunoglobulin polypeptide of thesame species, or an immunoglobulin or non-immunoglobulin polypeptide ofa different species.

As discussed in more detail elsewhere herein, antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, antibodies may be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins; see, e.g., international applicationsWO92/08495; WO91/14438; WO89/12624; U.S. Pat. No. 5,314,995; andEuropean patent application EP 0 396 387.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. Antibodies may be modified by natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theantibody, including the peptide backbone, the amino acid side-chains andthe amino or carboxyl termini, or on moieties such as carbohydrates. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given antibody. Also,a given antibody may contain many types of modifications. Antibodies maybe branched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicantibodies may result from posttranslation natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphatidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination;see, e.g., Proteins—Structure And Molecular Properties, T. E. Creighton,W. H. Freeman and Company, New York 2nd Ed., (1993); PosttranslationalCovalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990),626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

The present invention also provides for fusion proteins comprising anantibody, or antigen-binding fragment, variant, or derivative thereof,and a heterologous polypeptide. In one embodiment, a fusion protein ofthe invention comprises, consists essentially of, or consists of, apolypeptide having the amino acid sequence of any one or more of the VHregions of an antibody of the invention or the amino acid sequence ofany one or more of the VL regions of an antibody of the invention orfragments or variants thereof, and a heterologous polypeptide sequence.In another embodiment, a fusion protein for use in the diagnostic andtreatment methods disclosed herein comprises, consists essentially of,or consists of a polypeptide having the amino acid sequence of any one,two, three of the VH-CDRs of an antibody, or fragments, variants, orderivatives thereof, or the amino acid sequence of any one, two, threeof the VL-CDRs of an antibody, or fragments, variants, or derivativesthereof, and a heterologous polypeptide sequence. In one embodiment, thefusion protein comprises a polypeptide having the amino acid sequence ofa VH-CDR3 of an antibody of the present invention, or fragment,derivative, or variant thereof, and a heterologous polypeptide sequence,which fusion protein specifically binds to tau. In another embodiment, afusion protein comprises a polypeptide having the amino acid sequence ofat least one VH region of an antibody of the invention and the aminoacid sequence of at least one VL region of an antibody of the inventionor fragments, derivatives or variants thereof, and a heterologouspolypeptide sequence. In one embodiment, the VH and VL regions of thefusion protein correspond to a single source antibody (or scFv or Fabfragment) which specifically binds tau. In yet another embodiment, afusion protein for use in the diagnostic and treatment methods disclosedherein comprises a polypeptide having the amino acid sequence of anyone, two, three or more of the VH CDRs of an antibody and the amino acidsequence of any one, two, three or more of the VL CDRs of an antibody,or fragments or variants thereof, and a heterologous polypeptidesequence. In one embodiment, two, three, four, five, six, or more of theVH-CDR(s) or VL-CDR(s) correspond to single source antibody (or scFv orFab fragment) of the invention. Nucleic acid molecules encoding thesefusion proteins are also encompassed by the invention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84(1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531;Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA CellBiol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990),667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167);CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley etal., J. Exp. Med. 173 (1991),721-730); CTLA-4 (Lisley et al., J. Exp.Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991),1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991),2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991);and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991),Abstract No. 1448).

As discussed elsewhere herein, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be fused toheterologous polypeptides to increase the in vivo half life of thepolypeptides or for use in immunoassays using methods known in the art.For example, in one embodiment, PEG can be conjugated to the antibodiesof the invention to increase their half-life in vivo; see, e.g., Leonget al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002),531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

Moreover, antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be fused to marker sequences,such as a peptide to facilitate their purification or detection. Inparticular embodiments, the marker amino acid sequence is ahexa-histidine peptide (HIS), such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37 (1984), 767)and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart; see for example U.S. Pat. Nos. 5,116,964 and 5,225,538. The precisesite at which the fusion is made may be selected empirically to optimizethe secretion or binding characteristics of the fusion protein. DNAencoding the fusion protein is then transfected into a host cell forexpression.

Antibodies of the present invention may be used in non-conjugated formor may be conjugated to at least one of a variety of molecules, e.g., toimprove the therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy of the patient. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be labeled or conjugated either before or afterpurification, when purification is performed. In particular, antibodies,or antigen-binding fragments, variants, or derivatives thereof of theinvention may be conjugated to therapeutic agents, prodrugs, peptides,proteins, enzymes, viruses, lipids, biological response modifiers,pharmaceutical agents, or PEG.

Conjugates that are immunotoxins including conventional antibodies havebeen widely described in the art. The toxins may be coupled to theantibodies by conventional coupling techniques or immunotoxinscontaining protein toxin portions can be produced as fusion proteins.The antibodies of the present invention can be used in a correspondingway to obtain such immunotoxins. Illustrative of such immunotoxins arethose described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and byFanger, Immunol. Today 12 (1991), 51-54.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared e.g.by reacting a tau binding polypeptide with an activated ester of biotinsuch as the biotin N-hydroxysuccinimide ester. Similarly, conjugateswith a fluorescent marker may be prepared in the presence of a couplingagent, e.g. those listed herein, or by reaction with an isothiocyanate,or fluorescein-isothiocyanate. Conjugates of the antibodies, orantigen-binding fragments, variants or derivatives thereof of theinvention are prepared in an analogous manner.

The present invention further encompasses antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention conjugatedto a diagnostic or therapeutic agent. The antibodies can be useddiagnostically to, for example, demonstrate presence of a neurologicaldisease, to indicate the risk of getting a neurological disease, tomonitor the development or progression of a neurological disease, i.e.tauopathic disease as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment and/or prevention regimen.Detection can be facilitated by coupling the antibody, orantigen-binding fragment, variant, or derivative thereof to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions; see, e.g., U.S. Pat. No. 4,741,900 for metalions which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

An antibody, or antigen-binding fragment, variant, or derivative thereofalso can be detectably labeled by coupling it to a chemiluminescentcompound. The presence of the chemiluminescent-tagged antibody is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an antibody, or antigen-binding fragment,variant, or derivative thereof can be detectably labeled is by linkingthe same to an enzyme and using the linked product in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)” Microbiological Associates Quarterly Publication, Walkersville,Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol.31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio,E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980);Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo(1981). The enzyme, which is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibody, orantigen-binding fragment, variant, or derivative thereof, it is possibleto detect the antibody through the use of a radioimmunoassay (RIA) (see,for example, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,(March, 1986)), which is incorporated by reference herein). Theradioactive isotope can be detected by means including, but not limitedto, a gamma counter, a scintillation counter, or autoradiography.

An antibody, or antigen-binding fragment, variant, or derivative thereofcan also be detectably labeled using fluorescence emitting metals suchas 152Eu, or others of the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Techniques for conjugating various moieties to an antibody, orantigen-binding fragment, variant, or derivative thereof are well known,see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16(1985), and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev. 62 (1982), 119-158.

As mentioned, in certain embodiments, a moiety that enhances thestability or efficacy of a binding molecule, e.g., a bindingpolypeptide, e.g., an antibody or immunospecific fragment thereof can beconjugated. For example, in one embodiment, PEG can be conjugated to thebinding molecules of the invention to increase their half-life in vivo.Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54(2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

VI. Compositions and Methods of Use

The present invention relates to compositions comprising theaforementioned tau binding molecule, e.g., antibody or antigen-bindingfragment thereof of the present invention or derivative or variantthereof, or the polynucleotide, vector or cell of the invention. Thecomposition of the present invention may further comprise apharmaceutically acceptable carrier. Furthermore, the pharmaceuticalcomposition of the present invention may comprise further agents such asinterleukins or interferons depending on the intended use of thepharmaceutical composition. For use in the treatment of a tauopathicdisease, e.g., of the Alzheimer's disease the additional agent may beselected from the group consisting of small organic molecules, anti-tauantibodies, and combinations thereof. Hence, in a particular embodimentthe present invention relates to the use of the tau binding molecule,e.g., antibody or antigen-binding fragment thereof of the presentinvention or of a binding molecule having substantially the same bindingspecificities of any one thereof, the polynucleotide, the vector or thecell of the present invention for the preparation of a pharmaceutical ordiagnostic composition for prophylactic and therapeutic treatment of atauopathic disease, monitoring the progression of a tauopathic diseaseor a response to a tauopathic disease treatment in a subject or fordetermining a subject's risk for developing a tauopathic disease.

Hence, in one embodiment the present invention relates to a method oftreating a neurological disorder characterized by abnormal accumulationand/or deposition of tau in the brain and the central nervous system,respectively, which method comprises administering to a subject in needthereof a therapeutically effective amount of any one of theafore-described tau binding molecules, antibodies, polynucleotides,vectors or cells of the instant invention. The term “neurologicaldisorder” includes but is not limited to tauopathic diseases such asAlzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementiacomplex, argyrophilic grain dementia, British type amyloid angiopathy,cerebral amyloid angiopathy, corticobasal degeneration,Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillarytangles with calcification, Down's syndrome, frontotemporal dementia,frontotemporal dementia with parkinsonism linked to chromosome 17,frontotemporal lobar degeneration, Gerstmann-Sträussler-Scheinkerdisease, Hallervorden-Spatz disease, inclusion body myositis, multiplesystem atrophy, myotonic dystrophy, Niemann-Pick disease type C,non-Guamanian motor neuron disease with neurofibrillary tangles, Pick'sdisease, postencephalitic parkinsonism, prion protein cerebral amyloidangiopathy, progressive subcortical gliosis, progressive supranuclearpalsy, subacute sclerosing panencephalitis, tangle only dementia,multi-infarct dementia and ischemic stroke. Unless stated otherwise, theterms neurodegenerative, neurological or neuropsychiatric are usedinterchangeably herein.

A particular advantage of the therapeutic approach of the presentinvention lies in the fact that the antibodies of the present inventionare derived from B cells or B memory cells from healthy human subjectswith no signs of a tauopathic disease and thus are, with a certainprobability, capable of preventing a clinically manifest tauopathicdisease, or of diminishing the risk of the occurrence of the clinicallymanifest disease, or of delaying the onset or progression of theclinically manifest disease. Typically, the antibodies of the presentinvention also have already successfully gone through somaticmaturation, i.e. the optimization with respect to selectivity andeffectiveness in the high affinity binding to the target tau molecule bymeans of somatic variation of the variable regions of the antibody.

The knowledge that such cells in vivo, e.g. in a human, have not beenactivated by means of related or other physiological proteins or cellstructures in the sense of an autoimmunological or allergic reaction isalso of great medical importance since this signifies a considerablyincreased chance of successfully living through the clinical testphases. So to speak, efficiency, acceptability and tolerability havealready been demonstrated before the preclinical and clinicaldevelopment of the prophylactic or therapeutic antibody in at least onehuman subject. It can thus be expected that the human anti-tauantibodies of the present invention, both its target structure-specificefficiency as therapeutic agent and its decreased probability of sideeffects significantly increase its clinical probability of success.

The present invention also provides a pharmaceutical and diagnostic,respectively, pack or kit comprising one or more containers filled withone or more of the above described ingredients, e.g. anti-tau antibody,binding fragment, derivative or variant thereof, polynucleotide, vectoror cell of the present invention. Associated with such container(s) canbe a notice in the form prescribed by a governmental agency regulatingthe manufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition or alternatively the kit comprisesreagents and/or instructions for use in appropriate diagnostic assays.The composition, e.g. kit of the present invention is of courseparticularly suitable for the risk assessment, diagnosis, prevention andtreatment of a disorder which is accompanied with the presence of tau,and in particular applicable for the treatment of Alzheimer's disease(AD), amyotrophic lateral sclerosis/parkinsonism-dementia complex(ALS-PDC), argyrophilic grain dementia (AGD), British type amyloidangiopathy, cerebral amyloid angiopathy, corticobasal degeneration(CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuseneurofibrillary tangles with calcification, Down's syndrome,frontotemporal dementia, frontotemporal dementia with parkinsonismlinked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration,Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease,inclusion body myositis, multiple system atrophy, myotonic dystrophy,Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron diseasewith neurofibrillary tangles, Pick's disease (PiD), postencephaliticparkinsonism, prion protein cerebral amyloid angiopathy, progressivesubcortical gliosis, progressive supranuclear palsy (PSP), subacutesclerosing panencephalitis, tangle only dementia, multi-infarct dementiaand ischemic stroke.

The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, intranasal,topical or intradermal administration or spinal or brain delivery.Aerosol formulations such as nasal spray formulations include purifiedaqueous or other solutions of the active agent with preservative agentsand isotonic agents. Such formulations are adjusted to a pH and isotonicstate compatible with the nasal mucous membranes. Formulations forrectal or vaginal ad-ministration may be presented as a suppository witha suitable carrier.

Furthermore, whereas the present invention includes the now standard(though fortunately infrequent) procedure of drilling a small hole inthe skull to administer a drug of the present invention, in one aspect,the binding molecule, especially antibody or antibody based drug of thepresent invention can cross the blood-brain bather, which allows forintravenous or oral administration.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the dosage can range, e.g., fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg, or at least 1mg/kg. Doses intermediate in the above ranges are also intended to bewithin the scope of the invention. Subjects can be administered suchdoses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimesentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Progress can be monitored by periodic assessment.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents such as dopamine orpsychopharmacologic drugs, depending on the intended use of thepharmaceutical composition.

Furthermore, in a particular embodiment of the present invention thepharmaceutical composition may be formulated as a vaccine, for example,if the pharmaceutical composition of the invention comprises an anti-tauantibody or binding fragment, derivative or variant thereof for passiveimmunization. As mentioned in the background section, phosphor-tauspecies have been reported extracellularly in plasma and CSF (Aluise etal., Biochim Biophys. Acta. 1782 (2008), 549-558) and studies intransgenic mouse lines using active vaccination with phosphorylated taupeptides revealed reduced brain levels of tau aggregates in the brainand slowed progression of behavior impairments (Sigurdsson, J.Alzheimers Dis. 15 (2008), 157-168; Boimel et al., Exp Neurol. 224(2010), 472-485). Accordingly, it is prudent to expect that passiveimmunization with human anti-tau antibodies and equivalent tau bindingmolecules of the present invention would help to circumvent severaladverse effects of active immunization therapy concepts as alreadydiscussed in the background section. Therefore, the present anti-tauantibodies and their equivalents of the present invention will beparticularly useful as a vaccine for the prevention or amelioration oftauopathic diseases such as AD, ALS-PDC, AGD, CBD, CJD, FTD, FTDP-17,NP-C, PiD, PSP or other tauopathies as mentioned before.

In one embodiment, it may be beneficial to use recombinant bispecific ormultispecific constructs of the antibody of the present invention. For areference see Fischer and Léger, Pathobiology 74 (2007), 3-14. Suchbispecific molecule might be designed to target tau with one binding armand another pathologic entity such as Aβ or alpha-synuclein or adifferent pathological conformation of tau with a second binding arm.Alternatively the second binding arm may be designed to target a proteinpresent at the blood-brain-barrier to facilitate antibody penetrationinto the brain.

In one embodiment, it may be beneficial to use recombinant Fab (rFab)and single chain fragments (scFvs) of the antibody of the presentinvention, which might more readily penetrate a cell membrane. Forexample, Robert et al., Protein Eng. Des. Sel. (2008) October 16;S1741-0134, published online ahead, describe the use of chimericrecombinant Fab (rFab) and single chain fragments (scFvs) of monoclonalantibody WO-2 which recognizes an epitope in the N-terminal region ofAβ. The engineered fragments were able to (i) prevent amyloidfibrillization, (ii) disaggregate preformed Aβ1-42 fibrils and (iii)inhibit Aβ1-42 oligomer-mediated neurotoxicity in vitro as efficientlyas the whole IgG molecule. The perceived advantages of using small Faband scFv engineered antibody formats which lack the effector functioninclude more efficient passage across the blood-brain bather andminimizing the risk of triggering inflammatory side reactions.Furthermore, besides scFv and single-domain antibodies retain thebinding specificity of full-length antibodies, they can be expressed assingle genes and intracellularly in mammalian cells as intrabodies, withthe potential for alteration of the folding, interactions,modifications, or subcellular localization of their targets; see forreview, e.g., Miller and Messer, Molecular Therapy 12 (2005), 394-401.

In a different approach Muller et al., Expert Opin. Biol. Ther. (2005),237-241, describe a technology platform, so-called ‘SuperAntibodyTechnology’, which is said to enable antibodies to be shuttled intoliving cells without harming them. Such cell-penetrating antibodies opennew diagnostic and therapeutic windows. The term ‘TransMabs’ has beencoined for these antibodies.

In a further embodiment, co-administration or sequential administrationof other antibodies useful for treating a tauopathic disease may bedesirable. In one embodiment, the additional antibody is comprised inthe pharmaceutical composition of the present invention. Examples ofantibodies which can be used to treat a subject include, but are notlimited to, antibodies targeting beta-amyloid, alpha-synuclein, TDP-43and SOD-1.

In a further embodiment, co-administration or sequential administrationof other neuroprotective agents useful for treating a tauopathic diseasemay be desirable. In one embodiment, the additional agent is comprisedin the pharmaceutical composition of the present invention. Examples ofneuroprotective agents which can be used to treat a subject include, butare not limited to, an acetylcholinesterase inhibitor, a glutamatergicreceptor antagonist, kinase inhibitors, HDAC inhibitors,anti-inflammatory agents, divalproex sodium, or any combination thereof.Examples of other neuroprotective agents that may be used concomitantwith pharmaceutical composition of the present invention are describedin the art; see, e.g. international application WO2007/011907. In oneembodiment, the additional agent is dopamine or a dopamine receptoragonist.

A therapeutically effective dose or amount refers to that amount of theactive ingredient sufficient to ameliorate the symptoms or condition.Therapeutic efficacy and toxicity of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED50 (the dose therapeutically effective in 50% of thepopulation) and LD50 (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD50/ED50. In oneembodiment, the therapeutic agent in the composition is present in anamount sufficient to restore or preserve normal behavior and/orcognitive properties in case of AD, ALS-PDC, AGD, CBD, CJD, FTD,FTDP-17, NP-C, PiD, PSP or other tauopathic diseases as mentionedbefore.

From the foregoing, it is evident that the present invention encompassesany use of a tau binding molecule comprising at least one CDR of theabove described antibody, in particular for diagnosing and/or treatmentof a tauopathic disease as mentioned above, particularly Alzheimer'sdisease. In one embodiment, said binding molecule is an antibody of thepresent invention or an immunoglobulin chain thereof. In addition, thepresent invention relates to anti-idiotypic antibodies of any one of thementioned antibodies described hereinbefore. These are antibodies orother binding molecules which bind to the unique antigenic peptidesequence located on an antibody's variable region near theantigen-binding site and are useful, e.g., for the detection of anti-tauantibodies in sample of a subject.

In another embodiment the present invention relates to a diagnosticcomposition comprising any one of the above described tau bindingmolecules, antibodies, antigen-binding fragments, polynucleotides,vectors or cells of the invention and optionally suitable means fordetection such as reagents conventionally used in immuno or nucleic acidbased diagnostic methods. The antibodies of the invention are, forexample, suited for use in immunoassays in which they can be utilized inliquid phase or bound to a solid phase carrier. Examples of immunoassayswhich can utilize the antibody of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the radioimmunoassay (RIA), thesandwich (immunometric assay), flow cytometry and the Western blotassay. The antigens and antibodies of the invention can be bound to manydifferent carriers and used to isolate cells specifically bound thereto.Examples of well known carriers include glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amyloses, natural and modified celluloses, polyacrylamides, agaroses,and magnetite. The nature of the carrier can be either soluble orinsoluble for the purposes of the invention. There are many differentlabels and methods of labeling known to those of ordinary skill in theart. Examples of the types of labels which can be used in the presentinvention include enzymes, radioisotopes, colloidal metals, fluorescentcompounds, chemiluminescent compounds, and bioluminescent compounds; seealso the embodiments discussed hereinabove.

By a further embodiment, the tau binding molecules, in particularantibodies of the present invention may also be used in a method for thediagnosis of a disorder in an individual by obtaining a body fluidsample from the tested individual which may be a blood sample, a lymphsample or any other body fluid sample and contacting the body fluidsample with an antibody of the instant invention under conditionsenabling the formation of antibody-antigen complexes. The level of suchcomplexes is then determined by methods known in the art, a levelsignificantly higher than that formed in a control sample indicating thedisease in the tested individual. In the same manner, the specificantigen bound by the antibodies of the invention may also be used. Thus,the present invention relates to an in vitro immunoassay comprising thebinding molecule, e.g., antibody or antigen-binding fragment thereof ofthe invention.

In this context, the present invention also relates to meansspecifically designed for this purpose. For example, an antibody-basedarray may be used, which is for example loaded with antibodies orequivalent antigen-binding molecules of the present invention whichspecifically recognize tau. Design of microarray immunoassays issummarized in Kusnezow et al., Mol. Cell Proteomics 5 (2006), 1681-1696.Accordingly, the present invention also relates to microarrays loadedwith tau binding molecules identified in accordance with the presentinvention.

In one embodiment, the present invention relates to a method ofdiagnosing a tauopathic disease in a subject, the method comprisingdetermining the presence of tau and/or pathologically modified and/oraggregated tau in a sample from the subject to be diagnosed with atleast one antibody of the present invention, an tau binding fragmentthereof or an tau-binding molecule having substantially the same bindingspecificities of any one thereof, wherein the presence of pathologicallymodified and/or aggregated tau is indicative of a neurodegenerativetauopathy and an increase of the level of the pathologically modifiedand/or aggregated tau in comparison to the level of the physiologicaltau forms is indicative for progression of a neurodegenerative tauopathyin said subject.

The subject to be diagnosed may be asymptomatic or preclinical for thedisease. In one embodiment, the control subject has a tauopathicdisease, for example, AD, ALS-PDC, AGD, CBD, CJD, FTD, FTDP-17, NP-C,PiD, PSP or other tauopathies as mentioned before, wherein a similaritybetween the level of pathologically modified and/or aggregated tau andthe reference standard indicates that the subject to be diagnosed has atauopathic disease. Alternatively, or in addition as a second controlthe control subject does not have a tauopathic disease, wherein adifference between the level tau and/or of pathologically modifiedand/or aggregated tau and the reference standard indicates that thesubject to be diagnosed has a tauopathic disease. In one embodiment, thesubject to be diagnosed and the control subject(s) are age-matched. Thesample to be analyzed may be any body fluid suspected to containpathologically modified and/or aggregated tau, for example a blood, CSF,or urine sample.

The level tau and/or of pathologically modified and/or aggregated taumay be assessed by any suitable method known in the art comprising,e.g., analyzing tau by one or more techniques chosen from Western blot,immunoprecipitation, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), fluorescent activated cell sorting (FACS),two-dimensional gel electrophoresis, mass spectroscopy (MS),matrix-assisted laser desorption/ionization-time of flight-MS(MALDI-TOF), surface-enhanced laser desorption ionization-time of flight(SELDI-TOF), high performance liquid chromatography (HPLC), fast proteinliquid chromatography (FPLC), multidimensional liquid chromatography(LC) followed by tandem mass spectrometry (MS/MS), and laserdensitometry. In one embodiment, said in vivo imaging of tau comprisespositron emission tomography (PET), single photon emission tomography(SPECT), near infrared (NIR) optical imaging or magnetic resonanceimaging (MRI).

Methods of diagnosing a tauopathic disease such as Alzheimer's disease,monitoring a tauopathic disease progression, and monitoring a tauopathicdisease treatment using antibodies and related means which may beadapted in accordance with the present invention are also described ininternational applications WO93/08302, WO94/13795, WO95/17429,WO96/04309, WO2002/062851 and WO2004/016655. Similarly, antibody baseddetection methods for tau are described in international applicationWO2005/080986, the disclosure content of all being incorporated hereinby reference. Those methods may be applied as described but with a tauspecific antibody, binding fragment, derivative or variant of thepresent invention.

In a further aspect the present invention also relates to peptideshaving an epitope of tau specifically recognized by any antibody of thepresent invention. In one embodiment, such peptide comprises an aminoacid sequence as indicated in SEQ ID NO: 7, SEQ ID NO: 41, SEQ ID NO:42or a modified sequence thereof in which one, two, thre, four, five, six,seven or more amino acids are substituted, deleted and/or added, whereinthe peptide is recognized by any antibody of the present invention, forexample, by antibody NI-105.4E4 or NI-105.4E3.

In one embodiment of this invention such a peptide may be used fordiagnosing a neurodegenerative tauopathy in a subject, comprising a stepof determining the presence of an antibody that binds to a peptide in abiological sample of said subject, and being used for diagnosis of atauopathy in said subject by measuring the levels of antibodies whichrecognize the above described peptide of the present invention andcomparing the measurements to the levels which are found in healthysubjects of comparable age and gender. An elevated level of measuredantibodies specific for said peptide of the present invention would beindicative for diagnosing a tauopathy in said subject. The peptide ofthe present invention may be formulated in an array, a kit andcomposition, respectively, as described hereinbefore.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information and/or theNational Library of Medicine at the National Institutes of Health.Further databases and web addresses, such as those of the EuropeanBioinformatics Institute (EBI), which is part of the European MolecularBiology Laboratory (EMBL) are known to the person skilled in the art andcan also be obtained using internet search engines. An overview ofpatent information in biotechnology and a survey of relevant sources ofpatent information useful for retrospective searching and for currentawareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. Unlessotherwise stated, a term as used herein is given the definition asprovided in the Oxford Dictionary of Biochemistry and Molecular Biology,Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 019 850673 2. Several documents are cited throughout the text of thisspecification. Full bibliographic citations may be found at the end ofthe specification immediately preceding the claims. The contents of allcited references (including literature references, issued patents,published patent applications as cited throughout this application andmanufacturer's specifications, instructions, etc) are hereby expresslyincorporated by reference; however, there is no admission that anydocument cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLES

The examples which follow further illustrate the invention, but shouldnot be construed to limit the scope of the invention in any way. Thefollowing experiments in Examples 1 to 4 are illustrated and describedwith respect to antibodies NI-105.4E4, NI-105.24.B2 and 105.4A3 ascloned, i.e. the framework 1 (FR1) Ig-variable regions without beingadjusted to the germ line (GL) sequences of human variable heavy andlight chains; see FIG. 1.

Material and Methods

Detailed descriptions of conventional methods, such as those employedherein can be found in the cited literature; see also “The Merck Manualof Diagnosis and Therapy” Seventeenth Ed. edited by Beers and Berkow(Merck & Co., Inc. 2003).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. For furtherelaboration of general techniques useful in the practice of thisinvention, the practitioner can refer to standard textbooks and reviewsin cell biology and tissue culture; see also the references cited in theexamples. General methods in molecular and cellular biochemistry can befound in such standard textbooks as Molecular Cloning: A LaboratoryManual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); ShortProtocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley& Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985);Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization(Hames and Higgins eds. 1984); Transcription And Translation (Hames andHiggins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss,Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller andCalos, eds.); Current Protocols in Molecular Biology and Short Protocolsin Molecular Biology, 3rd Edition (Ausubel et al., eds.); andRecombinant DNA Methodology (Wu, ed., Academic Press). Gene TransferVectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold SpringHarbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.,eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In CellAnd Molecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir andBlackwell, eds., 1986). Protein Methods (Bollag et al., John Wiley &Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. eds.,Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., AcademicPress 1995); Immunology Methods Manual (Lefkovits ed., Academic Press1997); and Cell and Tissue Culture: Laboratory Procedures inBiotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents,cloning vectors and kits for genetic manipulation referred to in thisdisclosure are available from commercial vendors such as BioRad,Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech. General techniquesin cell culture and media collection are outlined in Large ScaleMammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997),148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); LargeScale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375);and Suspension Culture of Mammalian Cells (Birch et al., BioprocessTechnol. 19 (1990), 251); Extracting information from cDNA arrays,Herzel et al., CHAOS 11 (2001), 98-107.

Methods of Identification of Tau-Specific B-Cells and Cloning of theRespective Antibodies

Unless indicated otherwise below, identification of tau-specific B cellsand molecular cloning of anti-tau antibodies displaying specificity ofinterest as well as their recombinant expression and functionalcharacterization has been or can be generally performed as described inthe Examples and Supplementary Methods section of internationalapplication PCT/EP2008/000053 published as WO2008/081008, the disclosurecontent of which is incorporated herein by reference in its entirety. Anew method for identification of tau-specific B cells and molecularcloning of tau antibodies displaying specificity of interest as well astheir recombinant expression and functional characterization is providedwithin this application. As described above in one embodiment of thepresent invention cultures of single or oligoclonal B-cells are culturedand the supernatant of the culture, which contains antibodies producedby said B-cells is screened for presence and affinity of new anti-tauantibodies therein. The screening process comprises the steps of asensitive tissue amyloid plaque immunoreactivity (TAPIR) assay asdescribed in Example 1 and shown in FIG. 9; screen on brain extracts forbinding to PHFTau as described in Example 2 and shown in FIGS. 3 and 8;screening for binding of a peptide derived from tau of the amino acidsequence represented by SEQ ID NO:6 with phosphate groups on amino acidsSer-202 and Ser-205; on amino acid Thr-231; and/or on amino acidsSer-396 and Ser-404 of said sequence as analogously described in Example3 and shown in FIG. 5 with non-phosporylated peptides due to the epitopeconfirmation experiments for antibody NI-105.4E4; a screen for bindingof full-length tau of the amino acid sequence represented by SEQ ID NO:6and isolating the antibody for which binding is detected or the cellproducing said antibody as described in international patentWO2008/081008 and as described in Example 1 and shown in FIGS. 2, 5 and7.

Purification of Antigen

Recombinant human Tau40 was purchased from rPeptide (Bogart, Ga., USA).PHFTau was extracted from AD brain.

Isolation of paired helical filaments containing pathologicallyphosphorylated tau filaments (PHFTau) was performed following the methodby Goedert et al. (Goedert et al., Neuron 8 (1992), 159-168) withmodifications. One gram of AD brain tissue was cut into 5 mm pieces withall visible blood vessels removed. The tissue was washed with 40 ml icecold washing solution (100 mM Tris pH 7.4, 6 mM EGTA, 1 mM Na3VO4 and 1mM NaF) for three times followed by homogenization with 20 ml lysisbuffer (10 mM Tris pH 7.4, 0.8M NaCl, 1 mM EGTA, 1× protease inhibitorcocktail, 1 mM Na3VO4, 1 mM NaF, 1 mM AEBSF, 10% sucrose). Thehomogenate was centrifuged at 4° C. at 20′000×g for 20 mM Supernatantwas collected with addition of N-lauroyl sarcosinate (Sigma,Switzerland) to 1% (w/v). After two hours incubation at 37° C. withshaking, the supernatant was then centrifuged at 4° C. at 100′000×g forone hour. The pellet was collected and re-suspended in PBS. Afterclearing out possible contaminating immunoglobulins with protein Amagnetic beads, the PHFTau suspension was stored at −80° C. before use.A control extract from healthy control human brain tissue was preparedaccordingly.

Human Tau Antibody Screening ELISA:

96 well half area microplates (Corning) were coated with recombinant Tauprotein (rPeptide, Bogart, USA) at a standard concentration of 1 μg/mlin carbonate ELISA coating buffer (pH 9.6) overnight at 4° C. For PHFTauscreining, 96 well Immobilizer Microplates (Nunc, Denmark) were coatedwith PHFTau extracted from human AD brain at 1:100 dilutions incarbonate ELISA coating buffer (pH9.6) overnight at 4° C. Plates werewashed in PBS-T pH 7.6 and non-specific binding sites were blocked for 2hrs at RT with PBS-T containing 2% BSA (Sigma, Buchs, Switzerland). Bcell conditioned medium was transferred from memory B cell cultureplates to ELISA plates and incubated for one hour at RT. ELISA plateswere washed in PBS-T and then incubated with horse radish peroxidase(HRP)-conjugated donkey anti-human IgG (Fcγ fragment specific)polyclonal antibodies (Jackson immunoResearch, Newmarket, UK). Afterwashing with PBS-T, binding of human antibodies was determined bymeasurement of HRP activity in a standard colorimetric assay.

MULTI-ARRAY® Microplate Screening

Standard 96 well 10-Spot MULTI-SPOT plates (Meso Scale Discovery, USA)were coated with 30 ug/ml rTau (rPeptide), PHFTau brain extract andhealthy control brain extract in PBS. Non-specific binding sites wereblocked for 1 hr at RT with PBS-T containing 3% BSA followed byincubation with B cell conditioned medium for 1 hr at RT. Plates werewashed in PBS-T and then incubated with SULFO-Tag conjugated anti-humanpolyclonal antibody (Meso Scale Discovery, USA). After washing withPBS-T, bound of antibody was detected by electrochemiluminescencemeasurement using a SECTOR Imager 6000 (Meso Scale Discovery, USA).

Molecular Cloning of Tau Antibodies

Samples containing memory B cells were obtained from healthy humansubjects. Living B cells of selected memory B cell cultures areharvested and mRNA is prepared. Immunoglobulin heavy and light chainsequences are then obtained using a nested PCR approach.

A combination of primers representing all sequence families of the humanimmunoglobulin germline repertoire are used for the amplifications ofleader peptides, V-segments and J-segments. The first roundamplification is performed using leader peptide-specific primers in5′-end and constant region-specific primers in 3′-end (Smith et al., NatProtoc. 4 (2009), 372-384). For heavy chains and kappa light chains, thesecond round amplification is performed using V-segment-specific primersat the 5′-end and J-segment-specific primers at the 3′ end. For lambdalight chains, the second round amplification is performed usingV-segment-specific primers at the 5′-end and a C-region-specific primerat the 3′ end (Marks et al., Mol. Biol. 222 (1991), 581-597; de Haard etal., J. Biol. Chem. 26 (1999), 18218-18230).

Identification of the antibody clone with the desired specificity isperformed by re-screening on ELISA upon recombinant expression ofcomplete antibodies. Recombinant expression of complete human IgG1antibodies or chimeric IgG2a antibodies is achieved upon insertion ofthe variable heavy and light chain sequences “in the correct readingframe” into expression vectors that complement the variable regionsequence with a sequence encoding a leader peptide at the 5′-end and atthe 3′-end with a sequence encoding the appropriate constant domain(s).To that end the primers contained restriction sites designed tofacilitate cloning of the variable heavy and light chain sequences intoantibody expression vectors. Heavy chain immunoglobulins are expressedby inserting the immunoglobulin heavy chain RT-PCR product in frame intoa heavy chain expression vector bearing a signal peptide and theconstant domains of human immunoglobulin gamma 1 or mouse immunoglobulingamma 2a. Kappa light chain immunoglobulins are expressed by insertingthe kappa light chain RT-PCR-product in frame into a light chainexpression vector providing a signal peptide and the constant domain ofhuman kappa light chain immunoglobulin Lambda light chainimmunoglobulins are expressed by inserting the lambda light chainRT-PCR-product in frame into a lambda light chain expression vectorproviding a signal peptide and the constant domain of human or mouselambda light chain immunoglobulin.

Functional recombinant monoclonal antibodies are obtained uponco-transfection into HEK293 or CHO cells (or any other appropriaterecipient cell line of human or mouse origin) of an Ig- heavy-chainexpression vector and a kappa or lambda Ig-light-chain expressionvector. Recombinant human monoclonal antibody is subsequently purifiedfrom the conditioned medium using a standard Protein A columnpurification. Recombinant human monoclonal antibody can produced inunlimited quantities using either transiently or stably transfectedcells. Cell lined producing recombinant human monoclonal antibody can beestablished either by using the Ig-expression vectors directly or byre-cloning of Ig-variable regions into different expression vectors.Derivatives such as F(ab), F(ab)2 and scFv can also be generated fromthese Ig-variable regions.

Antibodies

Mouse monoclonal anti-human tau antibody Tau12 (Covance, Calif., U.S.A.)and mouse monoclonal tau antibody AT180 (Thermo Scientific, U.S.A.) wereused according to manufacturer's protocol. Recombinant human tauantibodies NI-105.4E4, NI105.24B2 and NI-105.4A3 are antibodies of thisinvention. They were expressed in HEK293 or CHO cells, purified fromconditioned media and were directly used in subsequent applicationsunless otherwise stated. In Examples 1 to 4 purified recombinantantibodies of the present invention were used.

Direct ELISA

96 well microplates (Costar, Corning, USA) were coated with recombinantTau protein (hTau40, rPeptide, Bogart, USA) diluted to a concentrationof 1 μg/ml in carbonate ELISA coating buffer (50 mM, pH9.6) at 4° C.over night. Non-specific binding sites were blocked for 2 hr at RT withPBS containing 2% BSA (Sigma, Buchs, Switzerland) and 0.5% Tween20.Binding of human antibodies of the present invention (NI-105.4E4,NI-105.24B2 and NI-105.4A3) was determined using HRP conjugated goatanti-human IgG Fcγ (Jackson immunoResearch, Newmarket, UK), followed bymeasurement of HRP activity in a standard colorimetric assay. EC50values were estimated by a non-linear regression using GraphPad Prismsoftware (San Diego, USA).

Western Blotting Protein Staining

PHFTau and recombinant hTau40 were resolved by gradient SDS-PAGE (NuPAGE4-12%; Invitrogen, Basel, Switzerland) followed by electroblotting onnitrocellulose membranes. After blocking the non-specific binding with2% BSA at room temperature for one hour, blots were incubated overnightwith primary antibodies NI-105.4E4, NI-105.24B2 (human) or Tau12 (mousemonoclonal antibody, Covance, Calif., U.S.A.), followed by aHRP-conjugated goat anti-human IgGFcγ (for human primary antibodies) ora HRP-conjugated goat anti-mouse IgG secondary antibody.

Blots were developed using ECL and ImageQuant 350 detection (GEHealthcare, Otelfingen, Switzerland).

PHFTau Extraction from AD Brain

Isolation of paired helical filaments containing pathologicallyphosphorylated tau filaments (PHFTau) was performed following the methodby Goedert et al. (Goedert et al., Neuron 8 (1992), 159-168) withmodifications. One gram of AD brain tissue was cut into 5 mm pieces withall visible blood vessels removed. The tissue was washed with 40 ml icecold washing solution (100 mM Tris pH 7.4, 6 mM EGTA, 1 mM Na3VO4 and 1mM NaF) for three times followed by homogenization with 20 ml lysisbuffer (10 mM Tris pH 7.4, 0.8M NaCl, 1 mM EGTA, 1× protease inhibitorcocktail, 1 mM Na3VO4, 1 mM NaF, 1 mM AEBSF, 10% sucrose). Thehomogenate was centrifuged at 4° C. at 20′000×g for 20 mM Supernatantwas collected with addition of N-lauroyl sarcosinate (Sigma,Switzerland) to 1% (w/v). After two hours incubation at 37° C. withshaking, the supernatant was then centrifuged at 4° C. at 100′000×g forone hour. The pellet was collected and resuspended in PBS. Afterclearing out possible contaminating immunoglobulins with protein Amagnetic beads, the PHFTau suspension was stored at −80° C. before use.A control extract from healthy control human brain tissue was preparedaccordingly.

Tau Peptides Synthesis

A peptide corresponding to amino acids 333-346 of hTau40(333GGGQVEVKSEKLDF346) which includes the epitope of NI-105.4E4identified by Pepspot mapping (amino acids 337-343) was synthesized bySchafer-N (Copenhagen, Denmark). An additional cysteine was added to theC-terminus to allow for covalent binding to Immobilizer Microplates(Nunc, Denmark). A second peptide corresponding to amino acids 226-239of human tau (226VAVVRpTPPKSPSSA239), the cognate epitope of thecommercially available mouse monoclonal tau antibody AT180 (ThermoScientific, USA) was synthesized accordingly and used as control.

Transgenic Mice

Three different animal models for tauopathies are used to validate thetau antibodies (and molecules with the binding specificities thereof) ofthe present invention.

1. Transgenic TauP301L mice (line183): expressing human Tau40 with P301Lmutation under the murine Thy1.2 promoter (Generation of thesetransgenic animals is described in Götz et al., J. Biol. Chem. 276(2001), 529-534 and in international application WO 2003/017918, thedisclosure content of which is incorporated herein by reference)

2. JNPL3 mice expressing the shortest human tau isoform with P301Lmutation under the murine PrP promoter (available from Taconic, Hudson,N.Y., U.S.A).

3. P301STau (line PS19) mice expressing human tau with P301S mutationunder the murine PrP promoter (available from the Jackson Laboratory,Bar Harbor, Me., U.S.A).

Tauopathies mouse models and corresponding wild type mice are kept understandard housing conditions on a reversed 12 h:12 h light/dark cyclewith free access to food and water. The treatment groups are balancedfor age and gender.

Example 1 Validation of Target and Binding Specificity of HumanTau-Antibodies

To validate tau as a recognized target of isolated antibodies directELISA assays were performed as described above. For the exemplaryrecombinant human NI-105.4A3 antibody 96 well microplates (Costar,Corning, USA) were coated with recombinant human tau (hTau40, rPeptide,Bogart, USA) diluted to a concentration of 3 μg/ml or with BSA incarbonate ELISA coating buffer (pH 9.6) and binding efficiency of theantibody was tested. The exemplary NI-105.4A3 antibody specificallybinds to human tau by ELISA. No binding is observed to BSA (FIG. 10).

For a determination of the half maximal effective concentration (EC50)of the exemplary antibodies NI-105.4E4 and NI-105.24B2 additional directELISA experiments with varying antibody concentrations were performed.96 well microplates (Costar, Corning, USA) were coated with recombinanthuman tau (hTau40, rPeptide, Bogart, USA) diluted to a concentration of1 μg/ml (for the assay with NI-105.4E4 Antibody), or of 3 μg/ml (for theassay with NI-105.24B2 Antibody) in carbonate ELISA coating buffer andbinding efficiency of the antibody was tested. The EC50 values wereestimated by a non-linear regression using GraphPad Prism software.Recombinant human-derived antibody NI-105.4E4 binds to hTau40 with highaffinity in the low nanomolar range at 2.4 nM EC50 (FIG. 2). NI-105.24B2binds to hTau40 with high affinity in the low nanomolar range at 6.6 nMEC50 (FIG. 7).

The half maximal effective concentration (EC50) of the exemplaryantibody NI-105.4A3 was also determined using direct ELISA experiments.ELISA plates were coated with recombinant human tau (hTau40, 1 ug/ml),PHFTau (1:100) and control preparation (1:100), and incubated withvarying antibody concentrations. NI-105.4A3 binds to rTau with highaffinity in the low nanomolar range at 1.4 nM EC50. NI-105.4A3 binds toPHFTau with high affinity in the low nanomolar range at 1.2 nM EC50(FIG. 12).

Example 2 Recombinant Human Antibodies Binding Analysis to RecombinantTau and Pathological Tau Extracted from AD Brain

To determine the binding capacity of NI-105.4E4 and NI-105.24B2 topathological tau species extracted from AD brain. SDS-PAGE and WesternBlot analysis was performed as described in detail above. Blots wereincubated overnight with primary antibodies NI-105.4E4 (human),NI-105.24B2 (human) or Tau12 (mouse monoclonal antibody, Covance,Calif., U.S.A.), followed by a HRP-conjugated goat anti-human IgGFcγ(for human antibodies) or a HRP-conjugated goat anti-mouse IgG secondaryantibody.

Recombinant antibodies NI-105.4E4 (FIG. 3) and NI-105.24B2 (FIG. 8)recognize recombinant hTau40 as well as pathologically modified PHFTauextracted from AD brain on Western blot. As expected, control antibodyTau12 recognizes both tau species as well (FIG. 3).

Additionally, as discussed in Example 1 above, the half maximaleffective concentration (EC50) of the exemplary antibody NI-105.4A3 wasdetermined in direct ELISA experiments using PHFTau. NI-105.4A3 binds toPHFTau with high affinity in the low nanomolar range at 1.2 nM EC50(FIG. 12).

Example 3 Mapping of the NI-105.4E4 and NI-105.4A3 Binding Epitope onhTau40

A peptide array of 118 peptide sequences covering the full-length hTau40(amino acids 1-441) with an overlap of 11 amino acids between twoadjacent peptides was spotted on a nitrocellulose membrane (JPT PeptideTechnologies GmbH, Berlin, Germany). Immunolabeling of antibodies aswell as membrane regeneration were carried out according tomanufacturer's instructions. To rule out non-specific binding of thedetection antibody, the membrane was first probed by HRP-conjugated goatanti-human IgG omitting the primary antibody (FIG. 4B). Afterregeneration the membrane was probed with 100 nM recombinant NI-105.4E4antibody. Bound antibody was detected using ECL and ImageQuant 350detection (GE Healthcare, Otelfingen, Switzerland).

Two groups of adjacent peptide spots (peptide 83, 84 and 85; peptide 96and 97) were specifically identified by NI105.4E4 (FIG. 4A), whencompared to the detection antibody only (FIG. 4B). The sequences coveredby these two groups of peptides correspond to amino acids 329-351 and387-397 of hTau40. These data suggest that NI-105.4E4 recognizes adiscontinuous epitope comprising two linear sequences: one within the R4microtubule binding domain and another in the C-terminal domain.

The sequence shared by peptides 83-85 comprises amino acid residues337-343 of hTau40. The Pepspot (JPT) data suggest that NI-105.4E4recognizes an epitope in hTau that comprises amino acids 337-343 ofhuman tau. This region is located within the microtubule binding domainof tau and is conserved among all neuronal human tau isoforms as well asacross other species including mouse and rat.

As this domain is bound to microtubules in physiologicalmicrotubule-associated tau, NI-105.4E4 is expected to preferentiallytarget the pathologically relevant pool of tau that is detached from themicrotubules.

To determine key residues within the NI-105.4E4 binding peptides, alaninscanning was performed to substitute each residue with alanine one at atime. The alanine residues in the original sequence (A384 and A390) weresubstituted to proline and glycine (FIG. 4E). Spots 35-50 and 51-68(FIG. 4C) are the original peptides (spot 35 and spot 51) and theiralanine substituted variants, whose amino acid sequences are shown inFIGS. 4D and E. Alanine scan suggests V339, E342, D387, E391 and K395are necessary for NI-105.4E4 binding.

An additional experiment has been performed by testing the binding ofNI-105.4E4 to tau peptides. Direct ELISA shows that NI-105.4E4specifically recognizes a peptide corresponding to amino acid 333-346 ofhTau40, which contains the amino acid residues 337-343 identified byPepspot mapping (FIG. 5). No cross-reactivity of NI-105.4E4 is observedto the control peptide covering the AT180 epitope. Vice versa, AT180recognizes its cognate epitope containing peptide but fails to bind tothe NI-105.4E4 specific peptide. Species-specific secondary antibodiesdo not bind to any of the peptides. Together, direct ELISA with coatedpeptides confirms that NI-105.4E4 specifically recognizes a peptidecontaining the amino acid residues 337-343 of human tau identified byPepspot mapping.

To grossly map the NI-105.4A3 binding epitope on hTau40, four tau domainpolypeptides (Tau domain I, domain II, domain III and domain IV) wereproduced. DNA fragments, synthesized using GeneArt® (Invitrogen), whichencode each Tau domain with 6× His tagged at the N-terminus were clonedinto the pRSET-A expression vector (Invitrogen), were transfected intoE. Coli BL21 (DE3) (New England Biolabs). The expressions of theHis-tagged Tau domains were induced by 0.5 mM IPTG for six hours beforebacteria were lysed with lysozyme with sonication. The lysate was boiledfor five minutes before being further purified with Ni-NTA SuperflowColumns (Qiagen). The eluted His-tagged Tau domains were coated on ELISAplates or loaded on polyacrylamide gel for Western Blot. Thesesequentially overlapping tau domain polypeptides cover the full lengthof hTau40 (FIG. 13A). Purified tau domains were coated on ELISA plateand the binding of NI-105.4A3 was tested. NI-105.4A3 binds only to taudomain I and the full length hTau40, indicating the epitope is withinthe N-terminal part of the hTau40 (aa1-136) (FIG. 13B). Western blotconfirms the specific binding of NI-105.4A3 to tau domain I (FIG. 13C).

NI-105.4A3 epitope mapping with PepSpot (JPT) technology identifiedamino acids Q35-Q49 of the human Tau40 (FIGS. 14A and C). To determinekey residues within the epitope for NI-105.4A3 binding, alanine scanningwas performed to substitute each residue with alanine one at a time. Thealanine residue in the original sequence (A41) was substituted withglycine and proline (FIG. 14B). Spots numbered from left to right with 1and 17 are the original epitope (spot 1) and its alanine substitutions,whose amino acid sequences are shown in FIG. 14C. Alanine scan showedthat D40, A41 and K44 are key residues for NI-105.4A3 binding.

Example 4 Assessment of the Binding of NI-105.4E4 to Physiological Formsas well as Pathological Aggregates of Tau AD Brain Tissues and in HumanTau Transgenic Mice

Neurofibrillary tangles (NFT) composed of hyperphosphorylated taufilaments are a neuropathological hallmarks of AD. Hyperphosphorylatedtau filaments are also the major components of dystrophic neurites andneuropil threads, both of which are common neuropathological features inAD. Overexpression of human tau containing the familial P301L taumutation in mice induces NFT formation at six months of age (Gotz etal., 2001a).

To assess the binding of recombinant human tau antibody to physiologicalforms as well as pathological aggregates of tau, immunohistologicalstainings were performed in AD brain tissues and in TauP301L transgenicmice with the exemplary NI-105.4E4 antibody of this invention.

Mice were perfused with 20 ml 100 mM TrisCl/6 mM EGTA (pH7.4) at roomtemperature under deep anesthesia. Brains were taken out and immersed in4% PFA in PBS (pH 7.4) at 4° C. over night for fixation followed byembedding in paraffin. For human tissue, paraffin blocks of braintissues from AD and healthy control subjects were used. DAB staining wascarried out following standard protocols. As positive control, mousemonoclonal antibody Tau-12 (Covance, Calif., U.S.A.) was used.HRP-conjugated detection antibodies without primary antibodies were alsoincluded.

Recombinant human antibody NI-105.4E4 identifies numerous NFTs andneuropil threads in AD brain (FIG. 6A), which are absent in healthycontrol brain (FIG. 6B). Secondary antibody alone does not give signalsin both AD (FIG. 6C) and control brain (FIG. 6D). In P301L tautransgenic mouse brain, NI-105.4E4 binds strongly to the pathologicaltau resembling NFT (FIGS. 6E, F and H), neuropil threads (FIGS. 6E andG) and dystrophic neurites (FIGS. 6E and H). In addition, NI-105.4E4also identifies tau aggregates at pre-tangle stage (FIG. 6I). In thebrain of transgenic mice overexpressing both human P301L tau and humanAPP with Swedish and Arctic mutations, NI-105.4E4 binds specifically todystrophic neurites surrounding beta-amyloid plaques (FIG. 6J).

Example 5 In Vivo Tests of the Antibodies of the Present Invention

As already described above studies in transgenic mouse lines usingactive vaccination with phosphorylated tau peptides revealed reducedbrain levels of tau aggregates in the brain and slowed progression ofbehavior impairments (Sigurdsson, J. Alzheimers Dis. 15 (2008), 157-168;Boimel et al., Exp. Neurol. 224 (2010), 472-485). However, activevaccination may not be particularly useable in humans because asignificant fraction of the elderly population is expected to benon-responders to vaccination. Furthermore, the potential side effectsassociated with a tau-directed immune response can be difficult tocontrol. Tau binding molecules of the present invention may bereasonably expected to achieve similar reductions in brain levels of tauaggregates as described above for the mouse antibodies, because of theirsimilar binding specificities against pathologically tau species.However, because of the evolutionarily optimization and affinitymaturation within the human immune system antibodies of the presentinvention provide a valuable therapeutic tool due to being isolated fromhealthy human subjects with high probability for excellent safetyprofile and lack of immunogenicity. Confirmation of these expectedtherapeutic effects may be provided by test methods as described in theabove mentioned experiments with mouse antibodies. In particular, theantibodies to be screened may be applied on diverse possible routes tothe animals such as intraperitoneal antibody injection, intracranialinjection, intraventricular brain infusion and tested for treatmenteffects. Either of the above mentioned application possibilities may bealso used after prior brain injection of beta-amyloid preparations intothe brain of tau transgenic mice to evaluate treatment effects on betaamyloid-induced tau pathology.

Evaluation of the treatment effects may be performed by histochemicalmethods comprising quantification of Gallyas positive cells counts,total human tau staining, brain burden of phosphorylated tau and/or abiochemical determination of brain soluble and insoluble tau andphosphor-tau levels upon sequential brain extraction. Further on,behavior testing of the treated mice may be performed, e.g., conditionedtaste aversion or contextual fear conditioning for a confirmation of thetherapeutic effects of the antibodies of the present invention(Pennanen, Genes Brain Behav. 5 (2006), 369-79, Pennanen Neurobiol Dis.15 (2004), 500-9.)

Example 6 Chimerization of Antibodies 4E4 and 4A3 with Mouse IgG2aConstant Domains

In order to generate antibodies with reduced immunogenicity for use inchronic treatment studies, mouse chimeric versions of antibodies 4E4 and4A3 were generated using recombinant DNA technology. A mouseIgG2a/lambda isotype was selected for these chimeric antibodies, inorder to generate a molecule which bound with high affinity to mouseFc-gamma receptors, and was therefore capable of inducing an immuneeffector response. The amino acid sequences of the chimeric 4E4 (ch4E4)and chimeric 4A3 (ch4A3) heavy and light chain constructs are shownbelow.

TABLE 3 Amino acid sequences of chimeric 4E4 (ch4E4 andchimeric 4A3 (ch4A3). mature ch4E4EVQLVESGGGLVQPGGSLKLSCAASGFNFNISAIHWVRQASGKGLEWVGR heavy chainIRSKSHNYATLYAASLKGRFTLSRDDSRNTAYLQMSSLQTEDMAVYYCTV (mouse IgG2a)LSANYDTFDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLV SEQ ID NO: 20KGYFPEPVTLIWNSGSLSSGVHTFPAVLQSDLYTLSSSVIVISSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVICVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVILTCMVIDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG K mature ch4E4SYELTQPPSVSVSPGQTARISCFGDTLPKQYTYWYQQKPGQAPVLVIYKD light chainTERPSGIPERFSGSSSGTIVILTISGVQAEDEADYYCLSADNSATWVFGG (mouse lambda)GTKVTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVIVDWK SEQ ID NO: 21VDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEG HTVEKSLSRADCS

Example 7 Chimerization of Antibodies 4E4 and 4A3 with Mouse IgG2aConstant Domains

A consensus N-linked glycosylation site was identified in the CDR1region of the 4E4 heavy chain. Upon mammalian (CHO) cell expression, thepredicted N-glycosylation site (Asn-30) was fully occupied by glycan, asdemonstrated by mass spectrometry. In order to eliminate N-glycosylationin this region and reduce product heterogeneity, Asn-30 of the heavychain of ch4E4 was changed to Gln (Table 4). When produced and purifiedfrom CHO cells, the modified antibody bound to recombinant tau with˜4-fold higher apparent binding affinity relative to the original,glycosylated antibody (see FIG. 15).

TABLE 4 Amino acid sequences of mature ch4E4(N30Q) heavy chain (mouseIgG2a). Substituted Gln residue is in bold, underlined. matureEVQLVESGGGLVQPGGSLKLSCAASGFNFQISAIHWVRQASGKGLEWVGR ch4E4 (N30Q)IRSKSHNYATLYAASLKGRFTLSRDDSRNTAYLQMSSLQTEDMAVYYCTV heavy chainLSANYDTFDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLV (mouse IgG2a)KGYFPEPVTLIWNSGSLSSGVHTFPAVLQSDLYTLSSSVIVISSTWPSQS SEQ ID NO: 22ITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVICVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVILTCMVIDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG K

Example 8 Production of Aglycosylated Chimeric 4E4 (ch4E4(N30Q) mIgG1Agly)

A mouse chimeric aglycosylated variant of 4E4 was produced (ch4E4(N30Q)IgG1-Agly) in order to evaluate the relationship between antibodyeffector function and activity. For the heavy chain, the variable domainof 4E4 were fused to a mouse IgG1 heavy chain containing an Asn to Glnmutation to eliminate the consensus Fc glycosylation site. The heavychain variable region also contained the N30Q change in order toeliminate the consensus N-glycosylation site in CDR1 (Example 7). Thelight chain was the ch4E4 lambda light chain described above.

Example 9 Acute Brain Penetration Study of Human 4E4 and 4A3

Human 4E4 and 4A3 were produced by transient transfection of CHO cellsand purified by affinity purification. The endotoxin levels werecontrolled and were all below 1 EU/mg. TauP301L mice wereintraperitoneally injected with 30 mg/kg 4E4 (n=7), 4A3 (n=7) antibodyor equal volume of PBS (n=7) at day 1 and day 4. At day 5, mice wereperfused under anesthesia with PBS containing 1 Unit/ml heparin. Blood,brain and spinal cord were collected for analyses. Right hemisphere ofthe brain was frozen at −80° C., left hemisphere of the brain and thespinal cord were post fixed in 10% neutralized formalin at 4° C. for twodays before being embedded in paraffin block and sectioned. Plasma wasstored at −80° C. in aliquots.

Brain protein extraction: frozen right hemisphere was weighed andhomogenized in 5 volumes (5 mL/g of wet tissue) of a solution containing50 mM NaCl, 0.2% diethylamine, protease inhibitors (Roche DiagnosticsGmbH) and phosphatase inhibitor (Roche Diagnostics GmbH). Samples werethen transferred to polycarbonate tubes and added another 5 volume ofhomogenization solution, and kept on ice for 30 mM. Soluble fraction wasthen collected after centrifugation at 100,000 g, 4° C. for 30 mM. Thissoluble fraction was used in human IgG assay. The pellet wasre-suspended in 3 volumes of PBS with protease and phosphataseinhibitor. After centrifugation at 16,000 g, 4° C. for 30 min,supernatants and pellets were stored separately at −80° C. for furtherinsoluble tau extraction. Pellets further extracted with modifiedsarcosyl extraction (Goedert M, Spillantini M G, Cairns N J, Crowther RA. Neuron 8, 159 (1992)).

Human IgG-specific sandwich ELISA: 2 μg/ml of goat anti-human IgG Fab(Jackson) in 50 mM carbonate ELISA coating buffer (pH9.6) was used ascapture antibody. Half-area 96-well microtitre plates was coated with 30μl/well with capture antibody at 4° C. over night. The plate was thenwashed 4 times with PBS containing 0.1% Tween 20 before incubating with50 μl/well PBS containing 2% BSA at room temperature for one hour.Soluble fractions of brain extracts, plasma samples and antibodystandard (4A3) were diluted in PBS containing 2% BSA and 0.1% Tween 20.30 μl of the diluted samples were added into each well and incubated atroom temperature for one hour. The plate was then washed with 200μl/well PBS containing 0.1% Tween 20 for four times before incubatedwith HRP-conjugated donkey anti-human Fcγ (Jackson, diluted at 1:10,000in PBS containing 2% BSA and 0.1% Tween 20) at room temperature for onehour. The plate was then washed with 200 μl/well PBS containing 0.1%Tween 20 for four times before adding 20 μl/well TMB (1:20 in 10 mMcitrate solution pH=4.1). The reaction was then stopped by adding 10 μl1M H2SO4 to each well. Antibody standard curve was obtained from serialdilutions of 4A3. Antibody concentrations in plasma and brain sampleswere calculated according to the standards. Brain human IgG level wasthen converted to ug antibody/gram fresh brain tissue (assuming 1 g/10ml) as indicated in FIG. 17.

High levels of human IgG were detected in the plasma of all 4E4 and 4A3treated mice. In contrast, no human IgG was detected in the plasma ofPBS treated mice (FIG. 16). Significant amount of human IgG was detectedin brain homogenates of 4E4 and 4A3 treated mice (FIG. 17).

Example 10 Chronic Study with Chimeric 4E4 and 4A3

Chimeric 4E4 and 4A3 containing the variable domains of the originalhuman antibody and the constant regions of mouse IgG2a may be producedby transient transfection of CHO cells and purified by affinitypurification. The endotoxin levels in each batch of the antibodies willbe controlled and kept below 1 Eu/mg. Gender balanced TauP301L mice atage of 7.5-8 months will be intraperitoneally injected with 10 mg/kg, 3mg/kg of antibody solution, or equal volume of PBS control. Eachtreatment group will have 20-25 mice. The treatment will be carried outonce a week for 26 weeks. Alternatively, the treatment will be carriedout twice a week for 13 weeks. Body weight will be monitored every twoweeks. Mice will be perfused under anesthesia at the end of thetreatment period. Brain, spinal cord and blood will be collected. Halfbrain and spinal cord may be post-fixed in 10% formalin for three daysbefore being embedded in paraffin block. 4-6 nm thick sections cut fromthese tissue blocks may be used for immunohistochemistry studies. Theother half brain will be weighted and deep frozen at −80° C. forbiochemical analyses.

Drug effects will be evaluated by comparing the level of neurofibrillarytangles (NFT) and the level of tau with different solubilitycharacteristics in treated and control samples. NFT may be visualized byGallyas silver impregnation (F Gallyas Acta Morphol. Acad. Sci. Hung19.1 (1971)), or by immunostaining with monoclonal mouse antibody AT100and AT180, which recognize pathologically phosphorylated tau in NFT. Thenumber or frequency of Gallyas-positive neurons and/or AT100, AT180labeled neurons in the brain and spinal cord in antibody treated miceand control animals may be determined to evaluate the effect of antibodytreatment.

Soluble and insoluble tau may be extracted following the brain proteinextraction protocol described herein. Alternatively, soluble andinsoluble tau may be extracted with modified sarcosyl extraction(Goedert M, Spillantini M G, Cairns N J, Crowther R A. Neuron 8, 159(1992)). Briefly, frozen brain is homogenized in 10 volumes (wt/vol) of10% sucrose homogenate buffer consisting of 10 mM Tris.HCl (pH 7.4), 0.8M NaCl, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, 1 mM AEBSF, proteaseinhibitors (Roche Diagnostics GmbH) and phosphatase inhibitor (RocheDiagnostics GmbH). The homogenate is spun for 20 mM at 20,000 g, and thesupernatant retained. The pellet is homogenized in 10 volumes ofhomogenization buffer and centrifuged for a second time. Thesupernatants may be pooled together, and N-lauryl-sarkosinate (Sigma) isadded to 1% (wt/vol) final concentration, and incubated at 37° C. with300 rpm rotation for 1.5 hour, followed by centrifugation at 100,000 gfor 1 h. The supernatant is collected as sarcosyl soluble fraction andthe pellet of 1 g brain tissue is re-suspended in 0.2 ml 50 mM Tris.HCl(pH 7.4) as PHF fraction.

The levels of soluble and insoluble tau will be measured withcommercially available Tau ELISA kits (Invitrogen). In addition, brainprotein extracts will be separated with 4-12% Bis-Tris SDS-PAGE followedimmunoblotting with Tau12 (human tau), AT8 (pS202/pT205), AT100(pT212/pS214), AT180 (pT231) and E178 (pS396) antibodies.Semi-quantitative analysis will be performed with measuring theintegrated density of each samples against standards of known quantitiesof tau.

Additionally, improvement of working memory in antibody treated mice canbe tested using a two-trial Y-maze task. The three arms of the maze are22 cm long, 5 cm wide and 15 cm deep. Black and white abstractive cluesare placed on a black curtain surrounding the maze. Experiments areconducted with an ambient light level of 6 lux during the dark phase.Each experiment comprises a training session and an observation session.During the training session, a mouse is assigned to two of the threearms (the start arm and the second arm), which can be freely exploredduring 4 min, with no access to the third arm (the novel arm). The mouseis then removed from the maze and kept in a holding cage for 1.5-5 min,while the maze is thoroughly cleaned with 70% ethanol to remove anyolfactory clues. The mouse is then put back again in the maze forobservation with all three arms accessible for 4 min. The number ofentry to each arm, the time spent in each arm, and the ratio of timespent in the novel third arm over the average of time spent in the othertwo arms (start arm and second arm) is calculated and compared amongdifferent treatment groups.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A human monoclonal anti-tau antibody, or a taubinding fragment thereof which (i) is capable of binding recombinanthuman tau; (ii) is capable of binding pathologically modified tau; (iii)binds to pathologically aggregated tau at the pre-tangle stage, inneurofibrillary tangles (NFT), neuropil threads and/or dystrophicneurites in the brain (iv) does not substantially bind to physiologicalforms of tau in the brain; (v) specifically binds any one of tauisoforms B to F or fetal tau represented by SEQ ID NOs: 1 to 6; (vi)specifically binds a tau C-terminus; (vii) specifically binds a tauN-terminus (viii) specifically binds a tau epitope located in themicrotubule binding domain which is masked in physiologicalmicrotubule-associated tau; (ix) specifically binds pathologicallyaggregated tau at the pre-tangle stage, in neurofibrillary tangles(NFT), neuropil threads and/or dystrophic neurites in the brain; (x)specifically binds a tau epitope which comprises the amino acid sequenceof SEQ ID NO: 7; (xi) specifically binds a tau epitope which comprisesthe amino acid sequence of SEQ ID NO: 41; (xii) specifically binds a tauepitope which comprises the amino acid sequences of SEQ ID NO: 7 and 41;or (xiii) specifically binds a tau epitope which comprises the aminoacid sequence of SEQ ID NO:
 42. 2. The antibody or tau binding fragmentthereof of claim 1 comprising: (a) a heavy chain CDR1 comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 23,29 and 35, a heavy chain CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:24, 30 and 36, and a heavy chainCDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 25, 31 and 37; (b) a light chain CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 26, 32 and 38, a light chain CDR2 comprising an amino acidsequence selected from the group consisting of SEQ ID NO:27, 33 and 39,and a light chain CDR3 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 28, 34 and 40; (c) a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 9, 13, 17 and 93; or (d) a light chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:11, 15 and
 19. 3. The antibody or taubinding fragment thereof of claim 2 which is a chimeric murine-human ora murinized antibody.
 4. An antibody or antigen-binding fragment thereofwhich competes with the antibody of claim 2 for specific binding to tau.5. The antibody or tau binding fragment thereof of claim 2, which isselected from the group consisting of a single chain Fv fragment (scFv),an F(ab′) fragment, an F(ab) fragment, and an F(ab′)2 fragment.
 6. Apolynucleotide encoding the antibody or tau binding fragment thereof ofclaim
 2. 7. A vector comprising the polynucleotide of claim
 6. 8. A hostcell comprising the vector of claim
 7. 9. A method for preparing ananti-tau antibody or tau binding fragment thereof, comprising (a)culturing the cell of claim 8; and (b) isolating said antibody or taubinding fragment thereof from the culture.
 10. An anti-tau antibody ortau binding fragment thereof obtainable by the method of claim
 9. 11.The anti-tau antibody or tau binding fragment thereof of claim 2, whichis (a) detectably labeled wherein the detectable label is selected fromthe group consisting of an enzyme, a radioisotope, a fluorophore and aheavy metal; or (b) which is attached to a drug.
 12. A compositioncomprising the anti-tau antibody or tau binding fragment thereof ofclaim 2, wherein the composition is (i) a pharmaceutical compositionfurther comprising a pharmaceutically acceptable carrier; or (ii) adiagnostic composition further comprising one or more reagentsconventionally used in immuno or nucleic acid based diagnostic methods.13. The composition of claim 12 further comprising an additional agentuseful for treating a neurodegenerative tauopathy.
 14. A method ofdiagnosing or monitoring the progression of a neurodegenerativetauopathy in a subject, the method comprising (a) assessing the level ofpathologically modified or aggregated tau in a sample from the subjectto be diagnosed with the antibody or tau binding fragment thereof ofclaim 2; and (b) comparing the level of modified or aggregated tau to areference standard that indicates the level of the pathologicallymodified or aggregated tau in one or more control subjects, wherein adifference or similarity between the level of pathologically modified oraggregated tau and the reference standard indicates that the subject hasa neurodegenerative tauopathy, wherein the neurodegenerative tauopathyis selected from the group consisting of Alzheimer's disease,amyotrophic lateral sclerosis/parkinsonism-dementia complex,argyrophilic grain dementia, British type amyloid angiopathy, cerebralamyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakobdisease, dementia pugilistica, diffuse neurofibrillary tangles withcalcification, Down's syndrome, frontotemporal dementia, frontotemporaldementia with parkinsonism linked to chromosome 17, frontotemporal lobardegeneration, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatzdisease, inclusion body myositis, multiple system atrophy, myotonicdystrophy, Niemann-Pick disease type C, non-Guamanian motor neurondisease with neurofibrillary tangles, Pick's disease, postencephaliticparkinsonism, prion protein cerebral amyloid angiopathy, progressivesubcortical gliosis, progressive supranuclear palsy, subacute sclerosingpanencephalitis, Tangle only dementia, multi-infarct dementia andischemic stroke.
 15. A method for in vivo detection of or targeting atherapeutic or diagnostic agent to tau in the human or animal body,comprising administering a composition comprising the antibody or taubinding fragment thereof of claim 2 attached to a therapeutic ordiagnostic agent, wherein said in vivo detection comprises positronemission tomography (PET), single photon emission tomography (SPECT),near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).16. A peptide having an epitope of tau specifically recognized by theantibody of claim 2, wherein the peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 9, 41, 42 andcombinations thereof.
 17. A method for diagnosing a neurodegenerativetauopathy in a subject, comprising detecting the presence of an antibodythat binds to the peptide of claim 16 in a biological sample of saidsubject.
 18. A kit useful in the diagnosis of a neurodegenerativetauopathy, said kit comprising the antibody or tau binding fragmentthereof of claim 2, with reagents or instructions for use.